Bnp(1-32) epitope and antibodies directed against said epitope

ABSTRACT

The present invention relates to a polypeptide carrying a human BNP(I-32) epitope according to Formula (I): a 1 -R 1 -X 1 -FGRKMDR-X 2 -R 2 -a 2  as well as ligands specific of the FGRKMDR epitope.

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/935,269, filed on Aug. 3, 2007, incorporated byreference herein.

The invention relates to human brain natriuretic peptides (BNP) and invitro diagnosis of congestive heart failure in humans. Moreparticularly, the invention relates to a new epitope present in theBNP(1-32) molecule, the antibodies directed against said epitope, inparticular the 20G7 monoclonal antibody, a method for the immunologicalassay of BNP(1-32) and proBNP(1-108) and respective fragments thereofusing such an antibody, and testing kits for carrying out said assays.

Congestive heart failure is a common clinical syndrome, in particularamong the elderly. It normally manifests itself in the form of aninsidious triggering of non-specific symptoms, such as coughing uponphysical exertion, fatigue and the appearance of peripheral oedema. Thediagnosis and assessment of the severity of the affection (graded instages I to IV NHYA in accordance with the New York Heart Association)are based on the combined interpretation of clinical signs and resultsof specific tests and examinations (echocardiography, scintigraphy,exercise test, etc.).

Due to the severity of congestive heart failure and also the highpatient care costs, early diagnosis of this syndrome is obviously highlydesirable as this would contribute to the implementation of treatmentssuitable for avoiding or delaying rapid progression of the syndrome tosevere congestive heart failure. It is therefore necessary to identifythose people at risk of congestive heart failure and/or unfavourableprognosis or subsequent complications. This would also make it possibleto propose the same tools for (quickly, simply and cost-effectively)therapeutically monitoring patients undergoing treatment. Nowadays, suchmethods for the diagnosis, prognosis and monitoring of congestive heartfailure are in place and are described below, but they have proved to besomewhat unsatisfactory and are not completely informative.

Acute coronary syndromes (ACS) are also a current major health problem.They comprise the following heart diseases: Q-wave myocardialinfarction, myocardial infarction with or without ST-segment elevation,threat of myocardial infarction or unstable angina.

The diagnosis, prognosis and monitoring of ACSs are also of the utmostimportance in the medical community. The assay of natriuretic peptides(BNP(1-32), NT-proBNP(1-76), and proBNP(1-108)) is of high interest inthese applications.

The same also applies to cases of dyspnoea (a disease characterised bybreathing difficulties), cerebrovascular accidents (CVA) (also known as“stroke” or “apoplexy”), and associated pathologies such as kidneyfailure and diabetis associated with these pathologies.

Presymptomatic markers which may predict congestive heart failure havelong been sought after. In this respect, it has been shown thatcardiomyocytes produce and secrete peptides with natriuretic activity: apeptide of auricular origin, ANP (Atrial Natriuretic Peptide) discoveredin rats by de Bold et al. Life Science 1981, vol. 28(1): 89-94, and anatriuretic peptide of auriculo-ventricular origin known as BNP (BrainNatriuretic Peptide) discovered by the inventors of patent EP 418 308and by Sudoh et al. (1988) Nature 332: 78-81 in pigs and in humans.

The precursor of BNP, preproBNP(1-134), is the form of storage of themolecule inside cardiomyocytes. Said precursor is cleaved during and/orafter secretion thereof in order to release a signal peptide andproBNP(1-108). ProBNP(1-108) consists in a polypeptide of 108 aminoacids of the sequence:H₁PLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPR₇₆S₇₇PKMVQGSGCFGRKMDRISSSSGLGCKV LRRH₁₀₈ (SEQ IDNO: 1).

It is cleaved before and/or during secretion thereof, between the Arg₇₆and Ser₇₇ amino acids into, on the one hand, BNP, also known asBNP(77-108) or BNP-32, or even BNP(1-32) (the term which will be usedhereinafter), and the N-terminal part of the prohormone, BNP(1-76), alsoknown as the N-terminal fragment of proBNP or NT-proBNP(1-76) (termwhich will be used hereinafter).

BNP(1-32), the vasoactive form of the molecule, consists in a peptide of32 amino acids of the sequence: S₁PKMVQGSGCFGRKMDRISSSSGLGCKVLRRH₃₂ (SEQID NO: 2)

17 amino acids form a loop closed by a disulphide bond between the twooxidised cysteine residues (C₁₀ and C₂₆), said loop being surroundedupstream by 9 amino acids (which constitute the N-terminal part), anddownstream by 6 amino acids (which constitute the C-terminal part).

The integrity of the loop is important for obtaining good biologicalactivity. Of the 17 residues forming the loop, 11 are also conserved inthe 2 other natriuretic peptides, which are ANP (A-type natriureticpeptide) and CNP (C-type natriuretic peptide).

NT-proBNP(1-76) is formed by the 76 N-terminal amino acids ofproBNP(1-108) and has the following sequence:H₁PLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPR₇₆ (SEQ ID NO: 3).

Interestingly, these 3 polypeptides, proBNP(1-108), NT-proBNP(1-76) andBNP(1-32), have proved to be good markers of congestive heart failure insuch a way that different assays with specific combinations ofantibodies have been developed.

Indeed, as proBNP(1-108) has been recognised as circulating in the bloodsince the initial studies of Tateyama et al. (1992) Biochemical andBiophysical Research Communications 185: 760-767, differentimmunological assays for detecting proBNP(1-108) have been suggested.Patent application WO 99/13331 thus describes a sandwich assay ofproBNP(1-108) with the aid of an antibody which recognises the 1-76portion of proBNP(1-108) and an anti-BNP(1-32) antibody. This type ofassay lacks sensitivity due to the binding, onto the solid phase(capture antibodies), of by-products of cleaved proBNP(1-108) having theepitope recognised by the capture antibody, that is to say,NT-proBNP(1-76) and the products resulting from the progressive cleavagethereof.

Application WO 2004/14952 describes the detection of proBNP(1-108) withthe aid, on the one hand, of an antibody which recognises an epitope ofthe sequence RAPRSP, located at the hinge of NT-proBNP(1-76) andBNP(1-32) (also called hinge 76 antibody) and, on the other hand, ananti-BNP(1-32) antibody in such a way that, in this type of assay, theassay is specific to the prohormone only, i.e. there is nocross-reactivity with the two forms of NT-proBNP(1-76) and BNP(1-32).

With regard to the assay of NT-proBNP(1-76), application WO 93/24531describes a method for in vitro diagnosis of congestive heart failurebased on the detection of NT-proBNP(1-76). However, the method describedin application WO 93/24531 does not appear to be easily carried out onNT-proBNP(1-76) in blood samples. Indeed, the only examples shown werecarried out, not on human sera, but on standard ranges obtained using asynthetic peptide, the peptide NT-proBNP(47-64). In order to overcomethis drawback, a highly sophisticated automated system has since provento be necessary.

Finally, a plurality of assays of BNP(1-32) have been developed usingvarious antibodies. For example, patent JP 2 676 114 B2 describes 2monoclonal antibodies (KY-hBNPI and KY-hBNPII) which recognise thecyclic structure of BNP(1-32), with no other details.

In addition, patent application EP 542 255 describes a monoclonalantibody which recognises the histidine H₃₂, last amino acid of theC-terminal K₂₇VLRRH₃₂ epitope of BNP(1-32). Other epitopes present onBNP(1-32) are known: 3 epitopes of the sequence ₁SPKMVQGSGC₁₀ (SEQ IDNO: 22), ₅VQGSGCFGR₁₃ (SEQ ID NO: 21), and ₁₅MDRISSSSGLG₂₅ (SEQ ID NO:23) are also described as being highly immunogenic in application WO97/32900 and U.S. Pat. No. 6,162,902. Said documents also describemonospecific antibodies directed against these epitopes.

In 2005, the HyTest company (Turku, Finland) put various antibodiesspecific of BNP(1-32) on the market. As described in the article bySeferian et al. (2007) Clinical Chemistry 53:5, 866-873, some of theseanti-BNP(1-32) antibodies target the 1-10, 11-22, 17-23 or 26-32 regionof BNP(1-32). Among the 17 antibodies obtained by immunising Balb/c miceusing the ₁₁FGRKMDRISSSS₂₂ (SEQ ID NO: 61) peptide of BNP(1-32), onlythe 24C5 and the 26E2 monoclonal antibodies are described. However,their epitopes are not precisely characterised: it is only indicatedthat they are directed against the above mentioned sequence of aminoacids 11 to 22 of BNP(1-32).

Patent application WO 2006/88700 describes another epitope present onBNP(1-32). It has the sequence of amino acids R₁₃(K₁₄)(M₁₅)D₁₆R₁₇I₁₈(SEQ ID NO: 24), included in the amino acids 13-20 which form part ofthe cyclic structure of human BNP(1-32). This application also describesa monoclonal antibody, designated 3-631-436, which recognises thisepitope specifically. The 4 amino acids R₁₃, D₁₆, R₁₇, and I₁₈ aredescribed as being functionally significant for binding the 3-631-436antibody to this epitope. The amino acids located upstream this epitope(such as phenylalanine F₁₁ and glycine G₁₂) are not mentioned.

One of the drawbacks of the BNP(1-32) hormone is that it is unstable inplasma and in serum. Indeed, protease-type enzymes seem to cleaveBNP(1-32). For example, Shimizu et al. (2002) Clinica Chimica Acta 316:129-135 report that the N-terminal part of BNP(1-32), more particularlythe Pro₂-Lys₃ bond, would be cleaved by proteases, as well as theArg₃₀-Arg₃₁ bond in the C-terminal position. Boerrigter et al. (2007)Am. J. Physiol. Regul. Integr. Comp. Physiol. 292: R897-90 and Hawkridgeet al (2005) Proc. Natl. Acad. Sci. USA. 102:17442-7 have described, inparticular, the degradation of BNP(1-32) in N-terminal position.

Likewise, some bond cleavages caused by endopeptidases have beenreported (Davidson & Struthers (1994) J. Hypertension 13: 329-336).Therefore, the assay of BNP(1-32) calls for specific precautions(Davidson et al (1995) Circulation 91:1276-7; Gobinet-Georges et al.(2000) Clin. Chem. Lab. Med. 38:519-23), and implies a suitable choiceof antibodies.

Recently, several teams have shown that natriuretic peptides couldcirculate in glycosylated and/or truncated form (Schellenberger (2006)Arch. Biochem. Biophys. 451: 160-6; Liang et al. (2007) Journal of theAmerican College of Cardiology 49: 1071-8; Lam et al. (2007) Journal ofthe American College of Cardiology 49: 1193-292).

With regard to early diagnosis of congestive heart failure, ACSs,dyspnoea and other cardiovascular diseases as well as CVAs andassociated pathologies, such as diabetis and kidney failure, there isalways a need to improve the reagents and methods for detectingBNP(1-32) and proBNP(1-108), in particular taking into account theproblem of the instability of BNP(1-32).

DESCRIPTION OF THE INVENTION

The present invention follows mainly from the entirely unexpectedfinding by the inventors of an unknown and unsuspected epitope whichexists in the human BNP(1-32) molecule and a specific monoclonalantibody which recognises said epitope. Contrary to all expectations,they discovered that the sequence F₁₁GRKMDR₁₇ (SEQ ID NO: 51) ofBNP(1-32) constitutes a beneficial epitope for generating antibodieswhich recognise the cyclic structure (amino acids 10-26) of BNP(1-32)and they obtained a monoclonal antibody, called 20G7, which specificallyrecognises said F₁₁G₁₂RK₁₄MDR₁₇ (SEQ ID NO: 51) epitope. In other words,the 20G7 antibody is a monoclonal antibody specific of the F₁₁GRKMDR₁₇(SEQ ID NO: 51) epitope of BNP(1-32).

The present invention also provides an immunoassay method for detectingBNP(1-32) and proBNP(1-108) as well as circulating fragments thereofusing the 20G7 monoclonal antibody and reagents containing saidantibody.

Indeed, the inventors noticed that, while synthesizing peptides locatedin the cyclic region (amino acids 10-26) of human BNP(1-32) andimmunising mice with said peptides, some resulting antibodies onlyreacted with peptides of this type if said peptides containedphenylalanine F₁₁ residues, lysine K₁₄ and Arginine R₁₇ and thatisoleucine I₁₈, which was particularly significant in the epitopedescribed in the international application WO2006/88700, did not belongto the epitope according to the present invention.

For example, the inventors immunised some mice with theTGCFGRKMDRISTSTAIGCKVL (SEQ ID NO: 4) peptide and others with theSGCYGRKMDRISTSTAIGCKVL (SEQ ID NO: 5) peptide. They observed that theimmune response to BNP(1-32) was much greater in the mice immunised withthe TGCFGRKMDRISTSTAIGCKVL (SEQ ID NO: 4) peptide than in the miceimmunised with the SGCYGRKMDRISTSTAIGCKVL (SEQ ID NO: 5) peptide. Itshould be noted that, in both cases, cysteines are presented in oxidisedform via an intrachain disulphide bond.

After lymphocytic fusion of immunised mice spleen cells with myelomacells, the inventors were thus able to produce different hybrid clones.In particular, they obtained a monoclonal antibody, called20G7-15/03/2007 (hereinafter, referred to as “20G7” for convenience),which only recognises those peptides of BNP(1-32) and proBNP(1-108)which contain the residues phenylalanine F₁₁, lysine K₁₄ and arginineR₁₇. In fact, these amino acids F₁₁, K₁₄ and R₁₇ have proven to beimportant for optimal binding of the 20G7 monoclonal antibody toBNP(1-32) and to proBNP(1-108) as well as to respective fragmentsthereof.

The hybridoma which secretes the 20G7-15/03/2007 (20G7) monoclonalantibody was deposited on Apr. 13, 2007 by Bio-Rad at the CNCM(Collection Nationale de Cultures de Microorganismes, Institut Pasteur,25, rue du Docteur Roux, 75 724 Paris Cedex 15, France) underregistration number CNCM I-3746.

Surprisingly and unexpectedly, the inventors observed with the 20G7antibody that, as soon as the residues F₁₁, K₁₄ and R₁₇ were substitutedby an alanine, either individually or jointly, the antigenic reactivityof the peptide, compared to that of the natural SGCFGRKMDRISSSSGLGCKVL(SEQ ID NO: 6) peptide, was considerably affected, whereas substitutingother amino acids of the epitope had almost no effect on the antigenicreactivity of the peptide. Also, an in-depth study on the 20G7 antibodyhas shown that it recognises the F₁₁GRKMDR₁₇ (SEQ ID NO: 51) epitope,but does not recognise the A₁₁GRKMDR₁₇ (SEQ ID NO: 62) sequence nor theGRKMDR₁₇I₁₈ (SEQ ID NO: 52) sequence, nor the C₁₀F₁₁GRKMD (SEQ ID NO:50) sequence.

The 20G7 monoclonal antibody thus recognises the F₁₁GRKMDR₁₇ (SEQ ID NO:51) epitopic sequence of BNP(1-32), but does not substantially recognisethe above mentioned VQGSGCFGR (SEQ ID NO: 21), SPKMVQGSGC (SEQ ID NO:22), and MDRISSSSGLG (SEQ ID NO: 23)) epitopes of application WO97/32900, nor the R(K)(M)DRI (SEQ ID NO: 24) epitope of applicationWO2006/88700.

The present invention therefore relates to a polypeptide carrying ahuman BNP(1-32) epitope having the formula (I):

a₁-R₁-X₁-FGRKMDR-X₂-R₂-a₂  (I)

wherein

a₁ may be H or represent a functional group or chemical group selectedfrom a thiol, alcohol, aminoxy, primary amine or secondary aminefunctional group, an amino carboxyl group, a biotinyl group and anacetyl group;

a₂ may represent an OH, NH₂ functional group or an alcoxyl group (aswill be clear for the man skilled in the art a₂ is attached to thecarbonyl (—CO—) moiety of the acidic function of the last amino acid ofthe polypeptide);

X₁ is absent or present and when present is selected among C and GC;

X₂ is absent or present and when present is selected among I and IS;

R₁ and R₂, which may be the same or different, absent or present,represent any amino acid or a peptide chain of 2 to 15 amino acids,provided that said polypeptide of formula (I) does not include anyportion of human BNP(1-32) of more than 11 amino acids including thesequence GCFGRKMDRIS (SEQ ID NO: 63).

As an equivalent alternative, formula (I) can also be defined asfollows:

a₁-(R₁)-(G)-(C)-FGRKMDR-(I)-(S)-(R₂)-a₂

wherein a₁, a₂, R₁ and R₂ are as defined above.

“Epitope” or “epitopic site” means an amino acid sequence which isrecognised by at least one antibody and allows the antibody to bindspecifically to said amino acid sequence.

In a preferred embodiment, R₁ and R₂ may be coupled to carriermolecules, reagents or marker molecules.

In another preferred embodiment, said polypeptide is selected in thegroup consisting of a₁-SGCFGRKMDR-a₂ (SEQ ID NO: 33), a₁-GCFGRKMDRI-a₂(SEQ ID NO: 34), a₁-CFGRKMDRIS-a₂ (SEQ ID NO: 35) and a₁-FGRKMDRISS-a₂(SEQ ID NO: 36), where a₁ and a₂ are as defined above.

In another preferred embodiment, the polypeptide as defined abovecorresponds to formula (II):

a₁-FGRKMDR-a₂  (II)

wherein a₁ and a₂ are as defined above.

The present invention also relates to the use of a polypeptide asdefined above, for the preparation of ligands directed against humanBNP(1-32) or human proBNP(1-108) as well as the respective fragmentsthereof comprising the sequence FGRKMDR (SEQ ID NO: 51).

“Fragment of proBNP(1-108)” according to the invention means anyfragment which is smaller than proBNP(1-108), including, in particular,BNP(1-32).

For example, the proBNP(3-108) fragment, as described in Lam et al.(2007) J. Am. Coil. Cardiol. 49:1193-1202 and produced by cleavage by adipeptidase. The term “fragment of proBNP(1-108)” according to theinvention also includes any polypeptide having been subjected to atleast one post-translational modification of proBNP(1-108), such asphosphorylation, glycosylation or the like. For example, Schellenbergeret al. (2006) Arch. Biochem. Biophys. 51:160-6 have shown thatproBNP(1-108) is a glycoprotein which is O-glycosylated either entirelyor in part.

“Fragment of BNP(1-32)” according to the invention means any fragmentwhich is smaller than BNP(1-32). In this case also, degradation ofBNP(1-32) has already been reported in the literature: for example, theBNP(3-32) fragment has been described by Lam et al (supra) and byHawkridge et al. (2005) Proc. Natl. Acad. Sci. USA 102:17442-7.

The terms “proBNP(1-108)” and “BNP(1-32)” as well as “fragment ofproBNP(1-108)” and “fragment of BNP(1-32)” also include any polypeptidehaving been subjected to at least one post-translational modification,such as phosphorylation, glycosylation or the like. For example,Schellenberger et al. (2006) Arch. Biochem. Biophys. 51:160-6 have shownthat proBNP(1-108) is a glycoprotein which is O-glycosylated eitherentirely or in part.

“Ligands directed against human BNP(1-32) or human proBNP(1-108) as wellas respective fragments thereof comprising the sequence FGRKMDR” refersto any molecule able to bind specifically to human BNP(1-32), to humanproBNP(1-108) or to fragments thereof.

The term “specific”, when it refers to recognition of a ligand orbinding of a ligand to a target, means that the ligand interacts withthe target without interacting substantially with another target whichdoes not structurally resemble the target. “Specific” recognition of theFGRKMDR epitope means that the interaction of the ligand with a targetcomprising the epitope does not substantially involve antigenicdeterminants, in particular amino acids, other than those of theepitope. In particular, this means that the ligand is able to bind asequence of amino acids of the BNP(1-32) and/or proBNP(1-108) sequenceas well as respective fragments thereof comprising the amino acidscomprising the FGRKMDR epitope, but is unable to bind a sequence ofamino acids of the BNP(1-32) and/or proBNP(1-108) sequence which doesnot comprise the FGRKMDR epitope in its entirety.

In addition, an amino acid present in an epitope is said to be“critical” as soon as its substitution by an alanine leads to areduction of at least 50% in the antigenicity of said epitope, accordingto Laune et al. (2002) Journal of Immunological Methods 267:53-70.

Moreover, an amino acid present in an epitope is said to be “essential”as soon as its substitution by an alanine leads to a reduction of atleast 80% in the antigenicity of said epitope.

Preferably, a ligand which specifically recognises the FGRKMDR epitopeaccording to the invention does not interact substantially with peptideshaving the sequence VQGSGCFGR (SEQ ID NO: 21), SPKMVQGSGC (SEQ ID NO:22), MDRISSSSGLG (SEQ ID NO: 23), RKMDRI (SEQ ID NO: 24) and RKMDRISS(SEQ ID NO: 25).

The expression “does not interact substantially with peptides having thesequence VQGSGCFGR, SPKMVQGSGC, MDRISSSSGLG, RKMDRI and RKMDRISS” meansthat the ligand has a cross reaction with one or other of thesesequences of less than 20%, preferably less than 10%, more preferablyless than 5%, particularly preferably less than 2%.

Within the scope of specific recognition of a target, binding constantsgreater than 10⁶ M⁻¹ are preferred, binding constants greater than 10⁸M⁻¹ are more preferred and binding constants greater than 10¹⁰ M⁻¹ areparticularly preferred.

Preferably, the residues F₁₁, K₁₄ and R₁₇ are also essential for bindinga ligand according to the invention to the epitope, since theirsubstitution by an alanine is characterised by a loss of 82%, 95% and85% respectively in the binding of the monoclonal antibody produced bythe hybridoma deposited on Apr. 13, 2007 by Bio-Rad at the CNCM(Collection Nationale de Cultures de Microorganismes, Institut Pasteur,25, rue du Docteur Roux, 75 724 Paris Cedex 15, France) underregistration number CNCM I-3746, to the epitopic peptide.

Preferably, the ligand is selected from the group constituted by anantibody or a fragment of said antibody which recognises the epitope, anaptamer, and a polypeptide which specifically recognises the epitopeobtainable by phage display.

In this context, the term “antibody” refers to any polyclonal ormonoclonal antibody.

The fragments scFv, Fab, Fab′, F(ab′)₂, as well as camelids single-chainantibodies are examples of antibody fragments which recognise theepitope.

The “aptamers” are well-known by the one skilled in the art. Aptamersare compounds of a nucleotide, in particular a ribonucleotide ordesoxyribonucleotide, or a peptide nature able to bind specifically to atarget, in particular a protein target. The aptamers of a nucleotidenature and the production thereof are described, in particular, byEllington et al. (1990) Nature 346:818-22 and Bock et al. (1992) Nature355:564-6. The aptamers of a peptide nature and the production thereofare described, in particular, by Hoppe-Seyler et al. (2000) J. Mol Med.78:426-30.

“Phage display” denotes a technique for selecting polypeptide ligandsexpressed on the capsid of a bacteriophage and encoded by a nucleicsequence inserted into the capsid encoding gene. This method is wellknown by the one skilled in the art and is described, in particular, byScott & Smith (1990) Science 249:386-390, and Marks et al. (1991) J.Mol. Biol. 222:581-597. Preferably, the polypeptide obtainable by phagedisplay is an scFv-type polypeptide (single-chain variable fragment).This technique is described, in particular, by Winter et al. (1994)Annu. Rev. Immunol. 12:433-455.

The ligands may also be obtained by chemical synthesis or by geneticengineering.

Preferably, the polypeptides as defined above are used to prepareantibodies, in particular monoclonal antibodies.

In this context, the invention also relates to use of a polypeptide asdefined above for the preparation of a hybridoma which secretes amonoclonal antibody directed against human BNP(1-32) or humanproBNP(1-108) as well as the respective fragments thereof comprising thesequence FGRKMDR.

The invention thus also relates to a method for preparing a hybridomawhich secretes a monoclonal antibody directed against human BNP(1-32) orhuman proBNP(1-108) as well as the respective fragments thereofcomprising the sequence FGRKMDR, wherein:

samples of lymphocytes secreting immunoglobulins are taken from ananimal, such as a mouse, rabbit or rat, immunised with a polypeptide asdefined above,

the lymphocytes are then fused with myeloma cells, such as Sp2 myelomacells (ATCC CRL-1581),

in order to obtain a hybridoma.

The present invention also relates to a hybridoma obtainable by themethod for preparing a hydridoma defined above.

More particularly, the present invention relates to the hybridomadeposited on Apr. 13, 2007 at the CNCM (Collection Nationale de Culturesde Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75 724Paris Cedex 15, France) under registration number CNCM I-3746.

Generally, the methodology used to obtain hybridomas and monoclonalantibodies may follow the conventional method of lymphocyte fusion andhybridoma culture described by Köhler & Milstein (1975) Nature256:495-497. Other methods for preparing monoclonal antibodies are alsoknown (e.g., Harlow et al, ed. 1988 “Antibodies: a laboratory manual”).

Alternative methods to this conventional method also exist. Monoclonalantibodies can be produced, for example, by expressing a nucleic acidcloned from a hybridoma.

The present invention also relates to a ligand specific of an epitope ofthe sequence FGRKMDR.

Preferably, the ligand is selected from the group constituted by anantibody or a fragment of said antibody which recognises the epitope, anaptamer, and a polypeptide which specifically recognises the epitopeobtained by phage display.

More preferably, the ligand is constituted by an antibody whichspecifically recognises an epitope of the sequence FGRKMDR, or afragment of said antibody which specifically recognises the epitope.Even more preferably, the ligand is constituted by a monoclonalantibody, in particular a monoclonal antibody produced by a hybridoma asdefined above.

The invention thus relates, in particular, to a ligand as defined above,constituted by the monoclonal antibody produced by the hybridomadeposited on Apr. 13, 2007 at the CNCM (Collection Nationale de Culturesde Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75 724Paris Cedex 15, France) under registration number CNCM I-3746.

The invention further more particularly relates to a ligand as definedabove harbouring at least one Complementary Determining Region (CDR), inparticular all the CDR, of the above-defined ligand constituted by themonoclonal antibody produced by the hybridoma deposited on Apr. 13, 2007at the CNCM (Collection Nationale de Cultures de Microorganismes,Institut Pasteur, 25, rue du Docteur Roux, 75 724 Paris Cedex 15,France) under registration number CNCM I-3746. CDR and methods fortransferring a CDR from an antibody to a ligand, preferably anotherantibody, are well known to the man skilled in the art and are notablydescribed e.g. in Nicaise et al. (2004) Protein Science 13:1882-1891 orKettleborough et al. (1991) Protein Engineering 4:773-783.

In addition, the ligands as defined above may be coupled to carriermolecules, reagents or labelled molecules.

The present invention also relates to a ligand as defined above fordetecting, in a biological sample, human BNP(1-32) or humanproBNP(1-108) as well as the respective fragments thereof comprising thesequence FGRKMDR.

In fact, the present invention also relates to a method for detecting,in a biological sample, human BNP(1-32) or human proBNP(1-108) as wellas respective fragments thereof containing the sequence FGRKMDR,comprising:

1) contacting the biological sample with at least one ligand as definedabove, preferably under conditions allowing the formation ofantigen-ligand complexes, and2) detecting any complexes which may have formed.

In the context of the invention, a “biological sample” or even a“biological fluid sample” is preferably constituted by a biologicalliquid, such as blood, plasma, serum, cerebrospinal fluid, saliva, urinaand lacrima, etc. (see e.g. Michielsen et al., (2008) Ann Clin Biochem45:389-94; Cortes et al., (2006) Eur J Heart Fail 8:621-7; Kirchhoff etal., (2006) J Neurotrauma 23:943-9; Kaneko et al., (1993) Brain Res612:104-9). As it is meant in this case, the term “biological sample”includes both the sample as taken and the sample which has beensubjected to various treatments, in particular to render it suitable foruse in the processes and methods according to the invention.

In a preferred embodiment, the above detection method comprises at leastone additional step of contacting the biological sample with at leastone additional ligand specific of human BNP(1-32) or human proBNP(1-108)and of the respective fragments thereof, with has a differentspecificity from that of the ligand according to the invention.

Preferably, the additional ligand is an antibody.

The present invention also relates to a method of diagnosis, prognosis,risk stratification or therapeutic follow-up of at least one cardiacand/or vascular pathology in an individual, comprising the followingsteps of:

1) contacting a biological sample from the individual with at least oneligand as defined above, preferably under conditions allowing theformation of antigen-ligand complexes,2) detecting any complex which may have formed, and,3) based on the result of the detection in step 2, determining adiagnosis, a prognosis, a risk of the development or therapeuticfollow-up of the pathology in the individual.

In a particular embodiment, the method defined above comprises at leastone additional step of contacting the biological sample with at leastone additional ligand specific of human BNP(1-32), or a humanproBNP(1-108) derivative, which has a different specificity to that ofthe ligand according to the invention.

Preferably, the additional ligand is an antibody.

Preferably, the pathology is selected from the group constituted of:

congestive heart failure,

acute coronary syndrome,

cerebrovascular accident,

kidney failure,

dyspnea,

high blood pressure,

atheromatous plaque rupture,

patent ductus arteriosus in premature newborns, and/or

diabetis.

“Congestive heart failure” means the pathological state in which ananomaly of the cardiac function is responsible for the heart beingunable to pump blood sufficiently to satisfy the metabolic needs of theorganism and/or in which the heart fulfils needs but with abnormallyhigh filling pressures. In particular, it may relate to a left and/orright ventricular failure.

“Acute Coronary Syndromes” denotes two categories in particular:

acute coronary insufficiency accompanied by a persistent upslope [i.e.elevation] of the ST segment revealing the formation of a Q-wavetransmural infarction corresponding generally to an acute total coronaryocclusion, and

acute coronary insufficiency with no upslope [i.e. with no elevation] ofthe ST segment corresponding to non-Q-wave infarction, also known asunstable anginas which correspond to plaque ruptures and incompletethromboses and require different treatment.

“Dyspnea” means the pathological state characterised by breathingdifficulties accompanied by feelings of obstruction or tightness. It isan extremely common symptom which may be due to several causes. Only amethodical approach enables appropriate treatment.

According to the definition of the World Health Organisation,“cerebrovascular accident” or “CVA” or “stroke” or “apoplexy” means thepathological state characterised by the rapid development of localisedor global clinical signs of cerebral dysfunction accompanied withsymptoms lasting more than 24 hours, which may result in death, with noapparent cause other than a vascular origin.

The process and the method above may be carried out in accordance withvarious formats well-known to the one skilled in the art, for example insolid or homogeneous phase, in one or two steps, by a sandwich method orby a competitive method.

Preferably, the sandwich method in solid phase between 2 ligands(preferably antibodies), one being a capture ligand and the other beinga detection ligand, will be used. This type of immunoassay isparticularly well-known to the one skilled in the art. For example, thearticle by Seferian et al. (2007) Clin. Chem. 53:866-873 gives anexample of a sandwich immunoassay (or immunometric assay at 2 sites) forassaying BNP(1-32) and proBNP(1-108), each time using a pair ofantibodies (an antibody immobilised in solid phase and an labelledantibody in detection).

“Capture ligand” means a ligand capable of binding the BNP(1-32) and/orproBNP(1-108) antigen, as well as the respective fragments thereof,present in the biological sample.

The presence of the antigen in the biological sample is revealed bydetection means, in particular a “detection ligand”. A detection ligand,which is labelled, is able to bind to the captured antigen, byrecognising an epitopic site which is different from that recognised bythe capture ligand.

The term “labelled” refers both to a direct labelling and to an indirectlabelling (for example, by means of other ligands, themselves directlylabelled, or using reagents of a labelled “affinity pair”, such as, butnot exclusively, the labelled avidin-biotin pair, etc.).

In the case of the sandwich method, the capture ligand is preferablyselected in such a way that it specifically recognises an epitope on thenatural antigen of the patient, whilst the detection ligand is selectedpreferably in such a way that it specifically recognises another epitopeon the natural antigen of the patient.

Preferably, the capture ligand is immobilised on a solid phase. By wayof non-limiting examples of solid phase, microplates could be used, inparticular polystyrene microplates, such as those sold by Nunc, Denmark.Solid particles or beads, paramagnetic beads, such as those produced byDynal, Merck-Eurolab (France) (under the trademark Estapor™) and PolymerLaboratories, or even polystyrene or polypropylene test tubes, glass,plastic or silicon chips, etc. may also be used.

ELISA assays, radioimmunoassays, or any other detection method may beused to reveal the presence of formed antigen-antibody complexes. Thus,different types of labelling of ligands in particular of antibodies, arepossible (radioactive, ezymatic, fluorescent, etc.).

The detection may also be carried out by new methods based on massaccumulation, such as surface plasmon resonance (SPR), by piezo-electricdetection, but also by mass spectrometry or any other methods defined asenabling the study of a ligand-antigen-type interaction in the absenceof a second labelled ligand.

A preferred implementation of the above process or method consists inusing a ligand as defined above immobilised on a solid phase incombination with at least one monoclonal or polyclonal antibody directedagainst the N-terminal portion of BNP(1-32), present in a labelled form.

Another preferred implementation of the above process or method consistsin using a ligand as defined above immobilised on a solid phase incombination with at least one monoclonal or polyclonal antibody directedagainst the C-terminal portion of BNP(1-32), such as the 50B7 antibody,(specific of the 26-32 peptide of BNP(1-32)), available from HyTest,present in a labelled form.

Alternatively, according to yet another preferred implementation of theabove method or process, a ligand as defined above may be used, in alabelled form, in combination with at least one monoclonal or polyclonalantibody directed against the N-terminal or C-terminal portion ofBNP(1-32), present in an immobilised form on a solid phase.

A preferred implementation of the above method or process consists inusing a ligand as defined above immobilised on a solid phase incombination with at least one monoclonal or polyclonal antibody directedagainst the N-terminal portion of NT-proBNP(1-108), such as the 16F3antibody, (specific of the 13-20 peptide of NT-proBNP), available fromHyTest, present a labelled form.

Alternatively, a preferred implementation of the above method or processconsists in using a ligand as defined above a labelled form incombination with at least one monoclonal or polyclonal antibody directedagainst the N-terminal portion of proBNP(1-108), such as the 16F3antibody, (specific of the 13-20 peptide of NT-proBNP), available fromHyTest, present in an immobilised form on a solid phase.

Another preferred implementation of the above method or process consistsin using a ligand as defined above immobilised on a solid phase incombination with a monoclonal antibody directed against the RAPR₇₆S₇₇P(SEQ ID NO: 55) sequence of proBNP(1-108) (such as the one described inpatent application WO2004/14952, or in Giuliani et al. (2006) Clin.Chem. 52:1054-1061), present in a labelled form.

Alternatively, a preferred implementation of the above method or processconsists in using a ligand as defined above in a labelled form incombination with a monoclonal antibody directed against the RAPR₇₆S₇₇P(SEQ ID NO: 55) sequence of proBNP(1-108) (such as the one described inpatent application WO2004/14952, or in Giuliani et al. (2006) Clin.Chem. 52:1054-1061), present in an immobilised form on a solid phase.

The present invention also relates to a multiepitopic calibrator havingthe following general formula (III):

t₁-E₁-L₁-E₂[-L_(k-1)-E_(k)]_(n)-t₂  (III)

wherein:

n is an integer between 0 and 8;

k is an integer between 3 and n+2 when n>0;

E₁, E₂, and E_(k) are different from one another, one representing aR₁-X₁-FGRKMDR-X₂-R₂ peptide sequence, wherein X₁, X₂, R₁ and R₂ are asdefined above, and the others representing a sequence of 3 to 15 aminoacids selected from the sequence of human proBNP(1-108);

t₁ represents a hydrogen atom, an acetyl group, a peptide sequence of 1to 10 amino acids, a peptide sequence of 1 to 10 N-α acetylated aminoacids, a biotinyl or biocytinyl group, a peptide sequence of 1 to 10amino acids carrying a biotinyl or biocytinyl radical, or a linear aminoalkyl (C₁-C₁₀) carbonyl chain;

t₂ represents a hydroxyl radical, an amino radical, a peptide sequenceof 1 to 10 amino acids, a peptide sequence of 1 to 10 amino acidscarrying a terminal amino group, or a linear or branched amino alkyl(C₁-C₁₀) carbonyl chain (as will be clear for the man skilled in the artt₂ is attached to the carbonyl (—CO—) moiety of the acidic function ofthe last amino acid of the E_(n) peptide chain);

L₁ and L_(k), which may be the same or different, represent a bindinggroup of peptide chains.

Preferably, the above multiepitopic calibrator corresponds to thefollowing general formula (IV):

t₁-E₁-L₁-E₂-L₂-E₃-t₂  (IV)

wherein E₁, E₂, E₃, L₁, L₂, t₁ and t₂ are as defined above.

Preferably, the above multiepitopic calibrator corresponds to thefollowing general formula (V):

t_(r)E₁-L₁-E₂-t₂  (V)

wherein E₁, E₂, L₁, t₁, and t₂, are as defined above.

The above standards (or calibrators) are used to establish standardcurves for the assays of BNP(1-32), proBNP(1-108) and/or one of theaforementioned fragments thereof. One advantage of said calibrators is,in particular, their stability.

Preferably, when used for assaying BNP(1-32), a biepitopic standardaccording to the invention comprises the FGRKMDR epitope according tothe invention and another, different epitope which is selected from thesequence of amino acids 77-108 of proBNP(1-108).

Preferably, when used for assaying proBNP(1-108), a biepitopiccalibrator according to the invention comprises the FGRKMDR epitopeaccording to the invention and another, different epitope which isselected from the sequence of amino acids 1-76 of proBNP(1-108) so as toensure the specificity of the proBNP(1-108).

Preferably, when used for assaying BNP(1-32), a triepitopic calibratoraccording to the invention comprises the R₁-X₁-FGRKMDR-X₂-R₂ epitopeaccording to the invention and two other, different epitopes which areselected from the sequence of amino acids 77-108 of proBNP(1-108).

Preferably, when used for assaying proBNP(1-108), a triepitopiccalibrator according to the invention comprises the R₁-X₁-FGRKMDR-X₂-R₂epitope according to the invention and two other, different epitopeswhich are selected from the sequence of amino acids of 1-108proBNP(1-108).

Preferably, in the above calibrator, R₁-X₁-FGRKMDR-X₂-R₂ represents apeptide sequence selected from the group constituted of SGCFGRKMDR (SEQID NO:33), GCFGRKMDRI (SEQ ID NO:34), CFGRKMDRIS (SEQ ID NO:35),FGRKMDRISS (SEQ ID NO:36), FGRKMDR (SEQ ID NO:8), SFGRKMDRISS (SEQ IDNO: 64), and CFGRKMDRISSSSGLGCK (SEQ ID NO: 65).

Preferably, the sequences different of R₁-X₁-FGRKMDR-X₂-R₂ are selectedfrom the group constituted by PRSPKMVQG (SEQ ID NO: 56), APRSPKMV (SEQID NO: 57), SGLGCKVL (SEQ ID NO: 58), SPKMVQGSG (SEQ ID NO: 59),YTLRAPRSPKMVG (SEQ ID NO: 60), YTLRAPRSPKMV (SEQ ID NO: 66),YTLRAPRSPKMVQG (SEQ ID NO: 67), SGLGCKVLRRH (SEQ ID NO: 68), andSGLGCKVLR (SEQ ID NO: 69).

Preferably, the multiepitopic calibrators according to the invention areselected from the group consisting of the multiepitopic calibratorsdefined by the following formulae:

Ac-YTLRAPRSPKMV-L₁-SFGRKMDRISS-NH₂;

Ac-YTLRAPRSPKMV-L₁-CFGRKMDRISSSSGLGCK-NH₂;

Ac-YTLRAPRSPKMVQG-L₁-FGRKMDR-NH₂;

Ac-FGRKMDR-L₁-SGLGC*KVLRRH-OH;

Ac-FGRKMDR-L₁-SGLGC*KVLR-NH₂;

Ac-SPKMVQGSG-L₁-FGRKMDR-NH₂;

Ac-YTLRAPRSPKMV-L₁-FGRKMDR-L₂-SGLGC*KVLRRH-OH;

and Ac-YTLRAPRSPKMV-L₁-FGRKMDR-L₂-SGLGC*KVLR-NH₂; wherein Ac representsan acetyl group, and C* represents an acetamidomethyl-blocked cysteine.

Preferably, L₁ and L₂ represent:

—NH—(CH₂)₅—CO—.

This group may, in particular, derive from the coupling agent known ashexanoic amino acid.

When E₁, E₂ and E₃ are present, the calibrator is said to betriepitopic, when only E₁ and E₂ are present, then the calibrator issaid to be biepitopic.

Moreover, the present invention also relates to a kit for detectinghuman BNP(1-32) or human proBNP(1-108) as well as the respectivefragments thereof comprising the sequence FGRKMDR, comprising at least:

a ligand as defined above; and

a multiepitopic calibrator as defined above and/or a polypeptide asdefined is above.

In a particular embodiment, the above kit also comprises a positivebiological control sample.

Preferably, the kit according to the invention comprises at least themonoclonal antibody produced by the hybridoma deposited on Apr. 13, 2007at the CNCM (Collection Nationale de Cultures de Microorganismes,Institut Pasteur, 25, rue du Docteur Roux, 75 724 Paris Cedex 15,France) under registration number CNCM I-3746.

The following examples and figures illustrate the invention, withoutlimiting it.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the reactivity of the 20G7 antibody with immobilisedpentadecapeptides representing the sequence of BNP(1-32) (from left toright, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 andSEQ ID NO: 17), synthesized by the Spot technique.

FIG. 2 shows the result of the alascan analysis of the binding of 20G7antibody to FGRKMDR epitope (substitution of each residue of the peptideby an alanine), and shows the importance of the F, K, and R residues.

FIG. 3 depicts the inhibition of the binding of antibodies 20G7 and 24C5to BNP(1-32) in the presence of increasing concentrations of solublepeptide of sequence SEQ ID NO: 51 (“AA11-AA17”, diamond), SEQ ID NO: 62(“mutated AA11-AA17”, circle) or SEQ ID NO: 9 (“deleted AA11-AA17”,triangle).

FIG. 4 shows the result of the alascan analysis of the binding of 11A8antibody to FGRKMDR epitope (substitution of each residue of the peptideby an alanine) and shows the importance of the F, G, K and R residues.

FIG. 5 represents a standard range of BNP(1-32) detected by the 20G7antibody.

FIG. 6 represents a standard range of recombinant proBNP(1-108) detectedby the 20G7 antibody.

FIG. 7 shows the correlation between the detection of BNP(1-32) andproBNP(1-108) by the 20G7 antibody in patients with congestive heartfailure.

FIG. 8 shows the correlation between the detection of BNP(1-32) andproBNP(1-108) by the 20G7 antibody in samples from subjects withcongestive heart failure of NYHA class I (FIG. 8A), NYHA class II (FIG.8B) and NYHA class III (FIG. 8C).

FIG. 9 shows the correlation between the detection of BNP(1-32) andproBNP(1-108) by the 20G7 antibody, in samples from healthy subjects.

FIG. 10 shows the correlation between the detection of BNP(1-32) andproBNP(1-108) by the 20G7 antibody in samples from subjects with renalfailure.

FIG. 11 shows BioPlex™ 2200 proBNP concentrations in ischemic stroke andcontrol citrated plasma samples. Notched box show the minimum, 25^(th),50^(th), 75^(th) percentiles and the maximum values.

FIG. 12 shows the correlation between the detection of BNP(1-32) andproBNP(1-108) by the 20G7 antibody in samples from subjects with acutecoronary syndrome.

FIG. 13 shows the correlation between the assay of glycosylatedproBNP(1-108) and the assay of unglycosylated proBNP(1-108) byimmunoassay using the immobilised hinge 76 antibody and in revelationthe 20G7 antibody according to the invention.

FIGS. 14 and 15 show a standard range of the biepitopic calibrators,CaliproBNP1 and CaliproBNP3, respectively, by using the Bioplex™ 2200device.

FIG. 16 shows a standard range of the biepitopic calibrator CaliproBNP5,proBNP(1-108) and BNP(1-32) by immunoassay using the immobilisedpolyclonal antibody L21016 and in revelation the 20G7 antibody accordingto the invention.

FIG. 17 shows a standard range of the triepitopic calibrator CaliproBNP6and proBNP(1-108) in two immunoassay formats: one based on theimmobilisation of the hinge 76 antibody and in revelation the 20G7antibody according to the invention (open and close circle), and thesecond one based on the immobilisation of the hinge 76 antibody and inrevelation an antibody directed against an epitope localised in theC-terminal part of the BNP(1-32) (open and close triangle).

EXAMPLES Example 1 Peptide Synthesis Materials and Methods:

Synthetic peptides were prepared by standard methods which are wellknown to the one skilled in the art. An example of this method isMerrifield synthesis, which is advantageous due to the fact that it canbe implemented easily (Merrifield, (1963); R. C. Sheppard (1971);Atherton et al. (1989)). “Pioneer” synthesisers from Perspective, or the“433A” synthesiser from ABI may be used as the automatic synthesiser.The peptides may also be obtained by homogenous phase synthesis.

The following syntheses were carried out in a Pioneer synthesiser using“Fmoc” chemistry (9-fluorenylmethyloxycarbonyl): in each step, thereagents (that is to say the protected amino acid and the couplingactivators (TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate)/HOBt (N-hydroxybenzotriazole)) were added in excess(in a “moles of reagent/moles of groups which can be substituted on theresin” ratio=5). At the end of the synthesis process, the peptide wasseparated from the resin by a trifluoroacetic acid solution (reagent K).The peptide was then precipitated in a cooled ether solution,lyophilised and then subsequently purified by HPLC.

In this way, the inventors synthesised peptides containing the followingamino acid sequences:

SEQ ID NO: 4: Ac-TGCFGRKMDRISTSTAIGCKVLCys-CONH2, SEQ ID NO: 5:5:Ac-SGCYGRKMDRISTSTAIGCKVL-CysCONH2 SEQ ID NO: 6:Ac-SGCFGRKMDRISSSSGLGCKVL-CysCONH2 SEQ ID NO: 7:Ac-SGCFGRKMDRIATSTAIGCKVL-CysCONH2 SEQ ID NO: 8:    Ac-FGRKMDR-CONH2SEQ ID NO: 9:     Ac-GRKMDR-CONH2 SEQ ID NO: 10:    Ac-FGRKMD-CONH2SEQ ID NO: 11:      Ac-RKMDRI-CONH2

Example 2 Immunogen Preparation: Coupling of a Peptide to a CarrierProtein for Immunisation

In order to immunise mice with these peptides, it is necessary to couplesaid peptides to a carrier protein such as KLH (keyhole limpethaemocyanin), thyroglobulin, or BSA (bovine serum albumin), viadifferent functional groups (thiol, amine, aldehyde, etc.) so as torender the peptide more immunogenic. The coupling reagent used to bindthe peptide to the protein may be heterobifunctional orhomobifunctional. The most frequently used reagents are BS3, sSMCC,SPDP, glutaraldehyde, etc.

The coupling method used involved the bifunctional sSMCC (Pierce,#22322) molecule, having an NHS ester functional group and a maleimidegroup as the chemical coupling agent, and KLH (Pierce, #77600) as thecarrier protein.

2-a. KLH Activation

Method:

20 mg of KLH were solubilised in 2 ml of phosphate buffered saline (20mM phosphate, 0.9 M NaCl pH 7.2) in order to obtain a finalconcentration of 10 mg/ml (do not vortex). In parallel, 4 mg of sSMCCwere solubilised with 400 μl of water for injection to obtain a finalconcentration of 10 mg/ml. 2 ml of KLH (20 mg) were subsequently mixedwith 200 μl of sSMCC (2 mg), and the mixture was incubated for 1 hour atroom temperature (20° C.) whilst being stirred slowly (20 revolutionsper min).

2-b. Desalting the Activated KLH:

Method:

A PD10 Sephadex TM G-25m column (Ge healthcare, USA, ref: 17-0851-01)was equilibrated with phosphate buffered saline (20 mM phosphate, 0.9 MNaCl pH 7.2, 100 mM EDTA). The 2 ml of activated KLH were deposited onthe column, and the elution was subsequently started with 3.5 ml of 20mM PBS buffer supplemented with 0.9 M NaCl, pH 7.2 and 100 mM EDTA; 500μl fractions were collected. The optical density (OD) was measured at280 nm for each fraction diluted to 1/25th and the fractions containingthe activated KLH were then identified and measured in accordance withthe Beer-Lambert law: OD=εCI: wherein OD is the optical density

ε=1.499,

C is the concentration and I=1 cm, the concentration of the activatedKLH may be determined and is reduced to 7.4 mg/ml in phosphate bufferedsaline.

2-c. Coupling of the Peptide to the Activated KLH

Method:

10 mg of lyophilised peptide were solubilised in 1 ml of Milli-Q water,which was degassed in an ultrasonic disintegrator to obtain a finalconcentration of 10 mg/ml, and were then mixed with 7.4 mg of activatedKLH (i.e. 1 ml of the solution obtained in 2-b.). This mixture was leftto incubate for 2 hours at room temperature (20° C.) whilst beingstirred slowly (20 rpm). A solution of cysteine at a concentration of 5mg/ml in a 20 mM PBS buffer+0.9 M NaCl pH 7.2 was subsequentlyintroduced to obtain a final concentration of 1 mM in the peptide/KLHsolution, and the entire mixture was left to incubate for 20 minutes atroom temperature (20° C.) whilst being stirred slowly (20 rpm).

2-d. Characterisation of the Coupled Peptide

Method:

The concentration of the coupled peptide was then determined by theBradford method (Bradford M., Anal. Biochem., 1976; 72: 248-54) asfollows: a standard range of from 50 to 1000 μg/mL of KLH was preparedin order to determine the KLH concentration of our sample from the OD at595 nm. In order to produce this standard range and to carry out thisassay, 50 μL of each point of the sample were diluted in 1.5 mL ofCoomassie blue (Bio-Rad, #1856210).

Having determined the concentration, PBS was added to the KLH-coupledpeptide to bring the concentration of the coupled peptide to 1 mg/mL.

Example 3 Immunisation of Mice and Production of Monoclonal Antibodies3-a) Immunisation of Mice:

In order to produce monoclonal antibodies, ten mice (Balb/c strainfemales, 5 weeks old, ref: SIFE055, Charles Rivers, Mass., USA) wereimmunised using one of the following peptides:

Ac-TGCFGRKMDRISTSTAIGCKVL-Cys-CONH2, (SEQ ID NO: 4)Ac-SGCYGRKMDRISTSTAIGCKVL-CysCONH2, (SEQ ID NO: 5)coupled to KLH in accordance with Example 2 (5 mice for each peptide).

For the first injection, an emulsion of 100 μg of KLH-coupled peptide(at a concentration of 1 mg/ml) diluted to ½ in Freund's completeadjuvant (Sigma, #F-5881) was prepared, and 200 μL of said emulsion(i.e. 100 μg of peptide) were injected subcutaneously into each mouse.At intervals of 20 days, three 200 μL booster shots of an emulsion ofKLH-coupled peptide (i.e. 100 μg of peptide) and Freund's incompleteadjuvant (Sigma, #F-5506) were injected subcutaneously, thenperitoneally, into each mouse.

20 days after the last booster shot, and after the antibodies obtainedhad been assessed by the ELISA method (in accordance with Example 4described below), the mouse with the greatest reaction against BNP(1-32)was retained in order to undergo hyperimmunisation, in accordance withthe following protocol:

-   -   subcutaneous injection of 200 μL of peptide-KLH at 1 mg/mL        diluted to 1/20^(th) with PBS    -   45 minute-wait    -   subcutaneous injection of 200 μL of peptide-KLH at 1 mg/mL        diluted to 1/20^(th) with PBS at a site different from the first        injection    -   45 minute-wait    -   subcutaneous injection of 200 μL of peptide-KLH at 1 mg/mL        diluted to 1/10^(th) with PBS at a site different from the        previous injections    -   30 minute-wait    -   intraperitoneal injection of 100 μL of promethazine (2.5%        Phenergan, injectable solution, UCB) diluted to 1 mg/mL with PBS    -   15 minute-wait    -   intraperitoneal injection of 200 μL of peptide-KLH at 1 mg/mL        diluted to 3/10^(ths) with PBS at a site different from the        previous injections

After these immunisations, the mouse S2 immunised with the SEQ ID NO: 4peptide was found to produce an antiserum which was very reactivetowards BNP(1-32) when using the protocol for detecting antibodiesdescribed below in Example 4. The lymphocytes from the spleen of saidmouse were subsequently subjected to lymphocyte fusion, carried out inaccordance with the protocol described below in 3b.

3-b) Production of Monoclonal Antibodies:

Lymphocyte fusion of the spleen cells of the immunised mouse S2 withmyeloma SP2 cells (ATCC CRL-1581) was carried out in accordance withKöhler and Milstein's well known protocol (1975) Nature 56:495-497.

The inventors were thus able to produce different hybrid clones. Inparticular, they obtained a monoclonal antibody, which was given thedesignation 20G7-15/03/2007 (referred to as “20G7” in the following forconvenience). The hybridoma which secretes the 20G7-15/03/2007 (20G7)monoclonal antibody was deposited at the CNCM (French NationalCollection of Cultures of Microorganisms, Institut Pasteur, 25, rue duDocteur Roux, 75 724 Paris Cedex 15, France) with the registrationnumber CNCM I-3746 on Apr. 13, 2007.

It goes without saying that other protocols for obtaining monoclonalantibodies which are well known to the one skilled in the art may beused.

Example 4 Detection of Anti-BNP(1-32) Antibodies to Assess the Responseof Mice During Immunisation 4.1 Materials:

The following reagents were used:

-   -   Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark)    -   PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets,        ref: 18912-014 (Invitrogen)    -   BNP(1-32): synthetic peptide (Sigma-Aldrich, USA, #B-5900)        or    -   proBNP(1-108) (recombinant protein produced in E. Coli, HyTest,        Finland)    -   Tween® 20 (Sigma-Aldrich, USA, #P1379)    -   anti-mouse IgG secondary antibody produced in rabbit and coupled        to peroxidase, (Sigma, USA, #A9044)    -   H₂O₂ (0.04% in 0.1 M citrate buffer, pH 4)    -   OPD (ortho-phenylenediamine, Sigma, USA, #P8412)    -   sulphuric acid (H₂SO₄, 4N)    -   serums from mice immunised in Example 3

4.2 Method and Principle:

An ELISA test was carried out on a solid support to detect the presenceof anti-BNP(1-32) antibodies in a mouse serum sample.

Some of the antigen was immobilised by adsorption in the cavities of a96-well microplate. After the remaining free sites were saturated andblocked, the immune serums were left to incubate, and the antibodies(<<Ac>>) which may have been present bound to the antigen (<<Ag>>) andformed an Ag-Ac complex. This complex was detected using animmunoconjugate (anti-mouse IgG antibody) coupled to an enzyme, which inthis case was HRP (horseradish peroxidase), which transforms acolourless substrate into a coloured product which indicates thepresence of the desired antibody. The formation of the final colouredproduct was quantified by carrying out an optical density reading at 490nm (OD). According to this method, which is well known to the oneskilled in the art, the OD obtained indicates the presence (high OD) orabsence (low OD) of antibodies in a tested mouse serum sample. There area number of variants of this test (antigen capture, competition assay .. . ) which are well known to the one skilled in the art.

1) Immobilisation of the Antigen on the Microplate:

Each antigen, BNP(1-32) or proBNP(1-108), was solubilised in PBS at afinal concentration of 0.5 μg/mL and was then immobilised, on the basisof 100 μL per well, on a Maxisorp microplate by being incubatedovernight at 4° C. After 3 washes with PBS 0.1% Tween® 20 (PBS-T), themicroplate was saturated with a solution (100 μL/well) of 0.1% PBS-Tcontaining 1% milk (semi-skimmed) and was then left to incubate at 37°C. for 1 hour.

2) Immunological Detection of the Antibodies Produced by the Mice:

The microplate was washed three times with 0.1% PBS-T. Each serum frompreviously immunised mice was subsequently diluted tenfold with 0.1%PBS-T containing 0.1% milk (semi-skimmed), then deposited on the basisof 100 μL per well and left to incubate for two hours at 37° C. Themicroplate was again washed three times with 0.1% PBS-T, then left toincubate for 1 hour at 37° C. in the presence of the conjugate coupledto peroxidase diluted to a 1/3,000^(th) in 0.1% PBS-T containing 0.1%milk (semi-skimmed) on the basis of 100 μL per well. Finally, themicroplate was washed three times with 0.1% PBS-T, then the peroxidasesubstrate was deposited on the basis of 100 μL per well. The microplatewas placed in darkness at room temperature for 20 minutes. The enzymaticreaction was stopped by adding 50 μL of sulphuric acid (H₂SO₄, 4 N) perwell, and the OD at 490 nm was subsequently measured in each well.

By using this method for detecting antibodies, the inventors found thatthe serum from the mouse S2 (immunised with the SEQ ID NO: 4 peptide)was very reactive with BNP(1-32), and proBNP(1-108). Following thelymphocyte fusion which was subsequently carried out between thelymphocytes from said hyperimmune mouse and the Sp2 myeloma, this methodalso made it possible to identify a hybridoma which produces animportant monoclonal antibody: the 20G7 hybridoma, producing the 20G7monoclonal antibody.

Example 5 Epitopic Characterisation of the 20G7 Monoclonal Antibody 5.1Epitopic Characterisation According to the <<Spot>> Technique 5.1.1Materials:

The equipment and reagents are all listed in C. Granier, S. Villard, D.Laune (Mapping and Characterization of Epitopes using the SPOT method.Cells/Cell Biology: A Laboratory Handbook, third edition (Volume 1),chapter 62, editor: Julio Celis, Elsevier, 2005).

5.1.2 Method:

The “SPOT” or “epitope mapping” method was used to characterise theepitope of the 20G7 monoclonal antibody. This method, described by Frank(Tetrahedron, 1992; 48: 9217-32), allows synthesis on a cellulosemembrane of a large number of peptides with sequences predetermined on afunctionalised support (aminopolyethyleneglycol-cellulose) and testingof their reactivity towards a soluble ligand, which is, in the presentcase, the 20G7 antibody.

5.1.2.1 Peptide Synthesis

The entire peptide synthesis process (amino acid activation, chemicalreaction, etc.) is detailed in Molina et al (Pept Res. 1996, Vol. 9: p151-5), and in C. Granier, S. Villard, D. Laune (Mapping andCharacterization of Epitopes using the SPOT method. Cells/Cell Biology:A Laboratory Handbook, third Edition (Volume 1), chapter 62, Editor:Julio Celis, Elsevier, 2005).

The BNP(1-32) sequence was synthesised entirely in the form ofoverlapping pentadecapeptides (SEQ ID NO: 12 to 20), with an offset oftwo amino acids:

SEQ ID NO: 12 SPKMVQGSGCFGRKM SEQ ID NO: 13    KMVQGSGCFGRKMDRSEQ ID NO: 14      VQGSGCFGRKMDRIS SEQ ID NO: 15        GSGCFGRKMDRISSSSEQ ID NO: 16          GCFGRKMDRISSSSG SEQ ID NO: 17           FGRKMDRISSSSGLG SEQ ID NO: 18              RKMDRISSSSGLGCKSEQ ID NO: 19                MDRISSSSGLGCKVL SEQ ID NO: 20                 RISSSSGLGCKVLRR

The other following peptides were also synthesised:

SEQ ID NO: 21 VQGSGCFGR SEQ ID NO: 22 SPKMVQGSGC SEQ ID NO: 23MDRISSSSGLG SEQ ID NO: 24 RKMDRI SEQ ID NO: 25 RKMDRISS

The BNP(1-32) sequence was also synthesised in the form of overlappingdecapeptides (SEQ ID NO: 26 to 48), with an offset of 1 amino acid:

SEQ ID NO: 26 SPKMVQGSGC SEQ ID NO: 27  PKMVQGSGCF SEQ ID NO: 28  KMVQGSGCFG SEQ ID NO: 29    MVQGSGCFGR SEQ ID NO: 30     VQGSGCFGRKSEQ ID NO: 31      QGSGCFGRKM SEQ ID NO: 32       GSGCFGRKMDSEQ ID NO: 33        SGCFGRKMDR SEQ ID NO: 34         GCFGRKMDRISEQ ID NO: 35          CFGRKMDRIS SEQ ID NO: 36           FGRKMDRISSSEQ ID NO: 37            GRKMDRISSS SEQ ID NO: 38             RKMDRISSSSSEQ ID NO: 39              KMDRISSSSG SEQ ID NO: 40              MDRISSSSGL SEQ ID NO: 41                DRISSSSGLGSEQ ID NO: 42                 RISSSSGLGC SEQ ID NO: 43                 ISSSSGLGCK SEQ ID NO: 44                   SSSSGLGCKVSEQ ID NO: 45                    SSSGLGCKVL SEQ ID NO: 46                    SSGLGCKVLR SEQ ID NO: 47                     SGLGCKVLRR SEQ ID NO: 48                      GLGCKVLRRHA selection of heptapeptides with an offset of one amino acid were alsosynthesised (SEQ ID NO: 49 to 53):

SEQ ID NO: 49 GCFGRKM SEQ ID NO: 50  CFGRKMD SEQ ID NO: 51   FGRKMDRSEQ ID NO: 52    GRKMDRI SEQ ID NO: 53     RKMDRIS

5.1.2.2 Immunological Test

The followed test for immunoreactivity has been described in detail inLaune et al (J. Immunol. Methods, 2002, Vol. 267(1), p 53-70). In short,the principle was as follows. The membrane was rehydrated by three TBSbaths (tris-buffered saline, pH 7.0) with a duration of 10 minutes ineach case, and was subsequently saturated by being incubated overnightat room temperature, whilst being stirred, in the presence of 15 ml of a10% saturation buffer (“blocking buffer”, Roche) and 5% saccharose inTBS 0.1% Tween® 20 (TBS-T). After the membrane was washed three timesfor 10 minutes with 0.1% TBS-T, the membrane was left to incubate for 90minutes at 37° C. whilst being stirred, in the presence of the antibodyto be tested (20G7 in this case) and the conjugate coupled to alkalinephosphatase diluted with the saturation buffer. After washing themembrane twice with 0.1% TBS-T, then twice with CBS (citrate bufferedsaline), each bath lasting 10 minutes, the alkaline phosphatasesubstrate was added and the membrane was incubated at room temperaturefor 1 to 30 minutes, depending on the speed at which the signalappeared.

5.1.2.3 Results

In the present case, the BNP(1-32) sequence was synthesised entirely inthe form of overlapping pentadecapeptides (SEQ ID NO: 12 to 20), with anoffset of two amino acids. As shown in FIG. 1, when the peptides arecontacted with the purified 20G7 antibody, only five successive peptidesreact with the antibody, and the common sequence thereof is F₁₁GRKMDR₁₇(FIG. 1):

SEQ ID NO: 13 K M V Q G S G C F G R K M D R SEQ ID NO: 14    V Q G S G C F G R K M D R I S SEQ ID NO: 15        G S G C F G R K M D R I S S S SEQ ID NO: 16            G C F G R K M D R I S S S S G SEQ ID NO: 17                F G R K M D R I S S S S G L G

To ensure that only this pattern is actually involved in the binding ofthe antibody to BNP(1-32), shorter peptides (decapeptides (SEQ ID NO: 26to 48) and heptapeptides (SEQ ID NO: 49 to 53) were also synthesised,with an offset of only one amino acid, in order to confirm and validatethe epitope. In each experiment, the common peptide sequence identifiedby 20G7 was F₁₁GRKMDIR₁₇ (for decapeptides, SEQ ID NO: 33 to 36, and forheptapeptides, SEQ ID NO: 51).

In order to determine which residues are critical and essential for therecognition of the epitope, each residue of the minimal sequenceF₁₁GRKMDR₁₇ was substituted successively by an alanine (A) in order toassess the involvement of each individual residue in accordance with the“Alascan” method, which is well known and described (Laune et al,above). As shown in FIG. 2, binding decreased to 82%, 95% and 85% whenthe F₁₁, K₁₄ and R₁₇ residues were respectively substituted by analanine, which indicates that these residues are essential.

These amino acids in positions 11, 14 and 17 are essential for therecognition of the epitope by the 20G7 monoclonal antibody. These dataare the mean values of a plurality (n=4) of repeat experiments. For thisreason, it is therefore clear that the F₁₁GRKMDIR₁₇ epitope, comprisingthe essential F₁₁, K₁₄ and R₁₇ amino acids, according to the invention,is different from that recognised in the patent application WO2006/88700which discloses another epitope (R₁₃(K₁₄)(M₁₅)D₁₆R₁₇I₁₈), the importantamino acids of which are R₁₃, D₁₆, R₁₇ and I₁₈.

In order to achieve a better understanding of the contributory effect ofthese residues to the binding process of the 20G7 antibody, the F₁₁, K₁₄and R₁₇ amino acids were substituted by amino acids with closedbiochemical properties. For example, F₁₁ was substituted by otheraromatic amino acids (tryptophan and tyrosine). The fact that thesequence composed of these “homologous” amino acids was recognised inthe same way by the 20G7 antibody suggests that it is the aromaticnature of the peptide in position 11 that is essential for the antibodybinding. With regard to K₁₄ and R₁₇, they were both substituted by anarginine and a lysine to study the effect of the lateral chain of theamino acid, and also the presence of a positive charge. It was alsofound in this case that the substitution was effectively conservative,since binding to 20G7 was retained, thus underlining the importance ofthe positive charge.

The same does not apply to the 24C5 antibody from HyTest, which hasdifferent essential residues.

The VQGSGCFGR, SPKMVQGSGC, MDRISSSSGLG, R₁₃KMDRI₁₈ and R₁₃KMDRISS₂₀ (SEQID NO: 21 to SEQ NO: 25) peptide sequences of human BNP(1-32), whichwere also synthesised on a membrane, were tested with the 20G7 antibodyusing the Spot method. Since residues which are essential for thebinding of 20G7 were absent, said antibody did not bind to said peptidesat all, with the result that 20G7 exhibited a cross reaction of lessthan 2% with these peptides.

5.2 Characterisation with Soluble Peptides

In a second step, to ensure that the F₁₁GRKMDR₁₇ peptide was really theepitope of the 20G7 antibody, this sequence was synthetized in a solubleform in order to carry out competition assays between this peptide andBNP(1-32). Parallely, in order to confirm the high contribution of theF₁₁ residue in the binding is to the 20G7 antibody, two other additionalpeptides (one wherein F was substituted by A, and the other wherein Fwas simply deleted) were also synthetised in a soluble form. Moreover,similar competition assays were carried out with the 24C5 antibody ofHytest to demonstrate that the importance of this residue is specific ofthe 20G7 antibody.

1—SEQ ID NO 51: sequence of the native epitope: F₁₁GRKMDR₁₇

2—SEQ ID NO 62: mutated sequence of the epitope: A₁₁GRKMDIR₁₇

3—SEQ ID NO 9: sequence deleted of the F₁₁ residue: G₁₂RKMDR₁₇

5.2.1 Materials

-   -   Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark)    -   PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets,        ref: 18912-014 (Invitrogen)    -   BNP(1-32): synthetic peptide (Sigma-Aldrich, USA, #B-5900)    -   Tween® 20 (Sigma-Aldrich, USA, #P1379)    -   monoclonal 20G7 antibody (Bio-Rad)    -   monoclonal 24C5 antibody (HyTest, Turku, Finland)    -   anti-mouse IgG secondary antibody produced in rabbit and coupled        to peroxidase, (Sigma, USA, #A9044)    -   H₂O₂ (0.04% in 0.1 M citrate buffer, pH 4)    -   OPD (ortho-phenylenediamine, Sigma, USA, #P8412)    -   sulphuric acid (H₂SO₄, 4N)

5.2.2 Methods

The principle of the immunoassay was the same as the one described in4.2. Briefly, synthetic BNP(1-32) was diluted in PBS buffer to bedirectly immobilised on a Maxisorp microplate at 0.5 μg/ml. A standardrange from 20 to 10,000 ng/ml of each soluble peptide AA11-AA17 (native,mutated or deleted) was prepared in buffer/serum and mixed to 100 μl ofmonoclonal antibody solution (20G7 or 24C5 antibody), at a finalconcentration of 0.5 μg/ml in PBS 0.1% Tween® 20 (PBS-T) containing 0.1%milk (semi-skimmed). The binding of the antibody was then detected by ananti-mouse conjugate labelled with peroxydase. The intensity of theresponse of the 20G7 antibody was compared to the one of monoclonal 24C5antibody from HyTest. The percentage of inhibition corresponding to thedecrease of the recognition of BNP(1-32) in the presence of the solublepeptide was determined for each soluble peptide, in order todetermine 1) that the sequence F₁₁GRKMDR₁₇ effectively represents the20G7 antibody epitope and 2) that the F₁₁ residue was essential for therecognition of BNP(1-32) by 20G7.

5.2.3 Results

FIG. 3 depicts the percentage of inhibition of the binding of themonoclonal antibody (20G7 or 24C5) to BNP(1-32) in the presence ofincreasing concentrations of soluble peptides (native: SEQ ID NO: 51,mutated: SEQ ID NO: 62, deleted: SEQ ID NO: 9).

It is highly remarkable to note the 20G7 antibody of the presentinvention behaves distinctly from the 24C5 antibody in the recognitionof BNP(1-32) in the presence of the soluble AA11-AA17 peptide mutated(SEQ NO:62) or deleted (SEQ NO:9). Adding soluble peptide mutated(A₁₁GRKMDR₁₇) or deleted (G₁₂RKMDR₁₇) does not inhibit the recognitionof BNP(1-32) by 20G7, whatever the added peptide concentration (up to 20μg/ml), whereas a total inhibition is observed when the native peptide(F₁₁GRKMDR₁₇ corresponding to the sequence SEQ ID NO: 51) is added. Thisexperiment confirms the importance of the F₁₁ residue in the binding ofthe 20G7 antibody to BNP(1-32), on the contrary to the 24C5 antibody.

Example 6 Epitopic Characterisation of Other Anti-BNP(1-32) MonoclonalAntibodies—Comparison with the 20G7 Antibody 6.1. Materials, Methods andProtocols:

The same nitrocellulose membranes and the same conditions of reactivityas those described in Example 5 were used to study these antibodies.

6.2. Results:

The inventors thus obtained a series of following characterizedmonoclonal antibodies: 20G7, 11A8, 17F10, Mab1, Mab2 and Mab3. As shownin Table 1, the other monoclonal antibodies have the same epitope as themonoclonal 20G7 of the present invention, i.e. FGRKMDR, but havedifferent essential amino acids compared to 20G7.

TABLE 1 Characteristics of the monoclonal antibodies MonoclonalEssential antibody Immunogen Epitope residues 20G7 SEQ ID NO: 4F₁₁GRKMDR₁₇ F, K and R 11A8 SEQ ID NO: 4 F₁₁GRKMDR₁₇ F, G, K and R 17F10SEQ ID NO: 4 F₁₁GRKMDR₁₇ F, K, D and R Mab1 SEQ ID NO: 4 F₁₁GRKMDR₁₇F, G, R and K Mab2 SEQ ID NO: 4 F₁₁GRKMDR₁₇ F and K Mab3 SEQ ID NO: 4F₁₁GRKMDR₁₇ F, G, R and K

FIG. 4 shows for example the result of the alascan analysis (successivesubstitution of each residue of the sequence F₁₁GRKMDR₁₇ by an alanineto assess the individual implication of each residue, method describedabove in 5.1.2.3) for 11A8 antibody. A substitution of the four residues(F, G, K and R) results in a significant loss of binding of the sequenceF₁₁GRKMDR₁₇, demonstrating that they are essential for the binding toBNP(1-32).

Example 7 Characterisation of the Antibody-Antigen Interaction of theMonoclonal Antibodies by Surface Plasmon Resonance Technology 7.1.Materials:

-   -   BIAcore® 2000 & 3000 analyser (Pharmacia, Uppsala, Sweden)    -   BNP(1-32) (synthetic peptide, Sigma, USA, #B-5900)    -   proBNP(1-108) (recombinant protein produced in E. Coli, HyTest,        Finland)    -   anti Fc fragment antibodies (Sigma, USA)    -   monoclonal antibodies 20G7, 11A8, 17F10 (Bio-Rad, Marnes la        Coquette, France)    -   PBS buffer (phosphate buffered saline), pH 7.4

7.2. Method: 7.2.1. Principle:

The BIAcore® 2000 & 3000 analyser (the principle of which is based onthe surface plasmon resonance technology (SPR)), was used to define thekinetics and the affinity of the interaction of the 20G7 monoclonalantibody and other monoclonal antibodies with BNP(1-32) orproBNP(1-108). The inventors followed the manufacturer's instructions.

The surface plasmon resonance SPR technique (BIAcore®, Pharmacia) wasdescribed in its entirety in Ferrières et al. (2000, FEBS Letters,479(3): 99-105). A monoclonal antibody was immobilised on a biosensor ora solid surface by using an anti Fc fragment antibody whilst the solubleantigen (BNP(1-32) or proBNP(1-108)) circulated at increasingconcentrations (0.001256 to 0.125 μg/ml) in a constant flow on thesurface of the biosensor at room temperature. The angle at which the SPRsignal is detected is directly proportional to the refractive index ofthe medium in which the evanescent wave propagates. The variations inthe refractive index are expressed in resonance units (RU, where 1000resonance units correspond to 1 ng of fixed proteins per mm² of activearea). The quantification of the interaction and the affinity betweenthe antigen and the monoclonal antibody is assessed by calculating theassociation rate constant (ka) and the dissociation rate constant (kd)by global data processing using the manufacturer's softwareBIAevaluation (BIAcore®, Pharmacia, Uppsala, Sweden). The equilibriumdissociation constant (KD=kd/ka) in mol/l reflects the affinity of theBNP(1-32) or proBNP(1-108) antigen for the monoclonal antibody.

7.2.2. Results:

Table 2 shows the characteristics of the interaction between themonoclonal anti-BNP antibodies (including 20G7) and the two recombinantantigens BNP(1-32) and proBNP(1-108).

TABLE 2 Interactions between various monoclonal anti-BNP antibodies andthe BNP(1-32) and proBNP(1-108) antigens. ka (M⁻¹ s⁻¹) kd (s⁻¹) KA (M⁻¹)KD (M) BNP 20G7 1.40 10⁶ 2.38 10⁻⁴ 5.90 10⁹ 1.70 10⁻¹⁰ 11A8 8.58 10⁵2.23 10⁻³ 3.85 10⁸ 2.59 10⁻⁹ 17F10 5.84 10⁵ 2.82 10⁻⁴ 2.07 10⁹ 4.8310⁻¹⁰ ProBNP 20G7 1.02 10⁶ 1.74 10⁻⁴ 5.90 10⁹ 1.69 10⁻¹⁰ 11A8 7.63 10⁵1.19 10⁻³ 6.43 10⁸ 1.56 10⁻⁹ 17F10 9.34 10⁵ 2.15 10⁻⁴ 4.35 10⁹ 2.3010⁻¹⁰

Table 2 summarises the different characteristics (association rateconstant (ka) and dissociation rate constant (kd) values allowing theequilibrium dissociation constant (KD in M) of the interaction betweenBNP(1-32) or proBNP(1-108) and the monoclonal antibodies to becalculated. These results for the interaction confirm the data obtainedwith the BNP(1-32) and proBNP(1-108) assays inasmuch as the 20G7monoclonal antibody exhibits an excellent association constant (ka) anda low dissociation constant (kd), allowing it to be characterised by anexcellent affinity constant of 1.70⁻¹⁰ M, identical for BNP(1-32) andproBNP(1-108) (Table 2).

Examples are also provided for other monoclonal antibodies (11A8 and17F10), the affinity constants of which are in the nanomolar range(2×10⁻¹⁰ to 9.35×10⁻¹⁰ M, Table 2).

Example 8 BNP(1-32) Assay Using the 20G7 Monoclonal Antibody 8.1.Materials:

-   -   Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark)    -   PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets,        ref: 18912-014 (Invitrogen)    -   BNP(1-32) synthetic peptide (Sigma-Aldrich, USA, #B-5900)    -   proBNP(1-108) (recombinant protein produced in E. Coli, HyTest,        Finland)    -   Tween® 20 (Sigma-Aldrich, USA, #P1379)    -   L21016 rabbit polyclonal antibody obtained by immunising rabbits        with an immunogen targeting the 1-10 region of BNP(1-32), its        epitope being the sequence S₁PKMV₅ (SEQ ID NO: 54) of BNP(1-32)    -   20G7 monoclonal antibody (Bio-Rad)    -   24C5 and 26E2 monoclonal antibodies (HyTest, Turku, Finland)    -   anti-mouse IgG antibody conjugate produced in rabbit and coupled        to peroxidase, (Sigma, USA, #A9044)    -   0.04% H₂O₂ in a 0.1 M citrate buffer, pH 4    -   OPD (ortho-phenylenediamine, Sigma, USA, #P8412)    -   sulphuric acid (H₂SO₄, 4N)

8.2. Method and Principle:

Initially, a standard range of 20 to 10,000 pg/ml of BNP(1-32) wasprepared in a buffer/serum from synthetic BNP(1-32).

The assay was based on the sandwich ELISA principle on a microplate,using the L21016 rabbit polyclonal antibody (Bio-Rad) for capture on asolid phase, its epitope being the sequence S₁PKMV₅ of BNP(1-32) fixedby passive adsorption by way of 100 μl of 5 μg/ml solution per well.

100 μl of monoclonal antibody solution (20G7, 24C5 or 26E2 antibodies)in solution at a concentration of 0.5 μg/ml in PBS 0.1% Tween® 20(PBS-T) buffer containing 0.1% milk (semi-skimmed) were used asdetection reagents. The intensity of the response of 20G7 was thuscompared to that of the 24C5 and 26E2 monoclonal antibodies.

Table 3 summarises the results of the analytical assay of BNP(1-32),expressed in optical density (OD) at 490 nm and obtained by saidantibodies in the presence of standard concentrations of BNP(1-32).

TABLE 3 OD values obtained during the analytical assay of BNP(1-32) withdifferent antibodies BNP (1-32) (pg/ml) 20G7 24 C5 26 E2 10,000 3.7520.077 0.048 5,000 3.056 0.068 0.041 2,500 1.950 0.067 0.035 1,250 1.1110.056 0.031 625 0.625 0.059 0.029 312.5 0.404 0.063 0.027 156.25 0.1800.071 0.032 78 0.099 0.052 0.055 39 0.066 0.055 0.032 20 0.031 0.0680.034 0 0.024 0.071 0.022

It is highly remarkable to note that the two antibodies 24C5 and 26E2behave quite differently from the 20G7 antibody of the presentinvention. These results thus confirm that in the latter assay format,20G7 is much more suitable than the 24C5 and 26E2 antibodies forBNP(1-32) assay.

The standard range shown in FIG. 5 and obtained with the 20G7 monoclonalantibody is linear from 20 to 10,000 pg/ml (r²=0.96). The two commercialantibodies 24C5 and 26E2 are not very effective or not at all effectivein detecting BNP(1-32), even at high concentrations of the analyte(Table 3).

Example 9 Study of the Complementarity of Monoclonal Antibodies in theSandwich ELISA 9.1. Materials:

-   -   Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark) primed        by the L21016 rabbit polyclonal antibody (Bio-Rad) which        recognises the sequence S₁PKMV₅ (SEQ ID NO: 54) of BNP(1-32)    -   PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets,        ref: 18912-014 (Invitrogen)    -   Tween®20 (Sigma-Aldrich, USA, #P1379)    -   BNP(1-32) synthetic peptide (Sigma-Aldrich, USA, #B-5900)    -   proBNP(1-108) (recombinant protein produced in E. Coli, HyTest,        Finland)    -   20G7 antibody (directed against the epitope F₁₁GRKMDR₁₇ (SEQ ID        NO: 8) prepared in different concentrations from 0.001 to 1        μg/ml    -   50B7 monoclonal antibody (HyTest, Finland) which recognises the        C-terminal portion of BNP(1-32) at a single concentration of 0.5        μg/ml    -   anti-mouse IgG secondary antibody produced in rabbit and coupled        to peroxidase, (Sigma, USA, #A9044)    -   0.04% H₂O₂ (in 0.1 M citrate buffer, pH 4, Sigma, USA)    -   OPD (ortho-phenylenediamine, Sigma, USA, #P8412)    -   sulphuric acid (H₂SO₄, 4N)

9.2. Method: 9.2.1. Principle:

The assay was based on the sandwich ELISA principle on a microplate,using the L21016 rabbit polyclonal antibody (Bio-Rad) for capture on asolid phase, its epitope being the sequence S₁PKMV₅ (SEQ ID NO: 54) ofBNP(1-32) fixed by passive adsorption (see Example 8), and a combinationof two monoclonal antibodies (the 20G7 monoclonal antibody directedagainst the epitope F₁₁GRKMDR₁₇ (SEQ ID NO: 8) and the 50B7 monoclonalantibody (HyTest, Finland) which targets the C-terminal portion ofBNP(1-32)) for detection. However, the 20G7 antibody was used atvariable concentrations while the 50B7 monoclonal antibody was used at aconstant concentration of 0.5 μg/ml.

The epitopic complementarity of the 20G7 and 50B7 antibodies was studiedat variable concentrations of the 20G7 antibody. This format allowed thecooperativity of the two monoclonal antibodies to be assessed in orderto improve the BNP(1-32) detection.

9.2.2. Protocol:

100 μl of 5 ng/ml BNP(1-32) solution was added into each microplatewell, in which the L21016 polyclonal antibody was adsorbed, and was leftto incubate for two hours at 37° C. The microplate was washed threetimes with 0.1% PBS-T, then 100 μl of a mixture containing the 50B7antibody and one of the 20G7 antibody dilutions were distributedthereon, and it was left to incubate for 2 hours at 37° C. After threewashes with 0.1% PBS-T, the peroxidase-rabbit anti-mouse IgG antibodyconjugate (diluted to a 1/3,000th with 0.1% PBS-T and containing 0.1%milk (semi-skimmed), on the basis of 100 μl per well, was left toincubate for 1 hour at 37° C. Finally, after 3 washes with 0.1% PBS-T,the H₂O₂+OPD solution was deposited on the basis of 100 μL/well. Themicroplate was placed in darkness at room temperature for 20 minutes.The enzymatic reaction was stopped by adding 50 μL of sulphuric acid(H₂SO₄, 4 N) per well, and the OD at 490 nm was subsequently measured ineach well.

9.2.3. Results

Table 4 shows the results of the analytical assay of BNP using the 20G7and 50B7 monoclonal antibodies, expressed in optical density at 490 nm.

TABLE 4 Cooperativity of the two monoclonal antibodies for detectingBNP(1-32) 20G7 range Optical Density (μg/ml) 490 nm 0.5 3.792 0.1 3.7530.05 3.747 0.01 3.531 0.005 3.272 0.001 1.948 0 1.296

It was noted that there is synergism between the two antibodies. Inother words, it was noted that the effects of the two antibodies wereadded: in the absence of 20G7, the signal (OD) was limited to 1.296, andthe more 20G7 was added, the more the resulting signal (OD) increased.

By using both the 20G7 monoclonal antibody, which targets theF₁₁GRKMDR₁₇ (SEQ ID NO: 8) epitope according to the invention (locatedin the loop of BNP(1-32)), and the 50B7 monoclonal antibody, whichtargets the C-terminal region of BNP(1-32), BNP(1-32) detection issignificantly improved. This demonstrates the cumulative or cooperativecontribution of the two monoclonal antibodies used in detection.

This kind of complementarity may also be envisaged between the 20G7monoclonal antibody and other antibodies which recognise an epitopelocated in other positions in the BNP(1-32) sequence (principally in theN-terminal position, C-terminal position). The number of antibodies usedmay also be greater than two as long as no steric hindrance problems areencountered.

Example 10 ProBNP(1-108) Assay Using the 20G7 Monoclonal Antibody 10.1.Materials:

-   -   BNP(1-32) synthetic peptide (Sigma-Aldrich, USA, #B-5900)    -   proBNP(1-108) (recombinant protein produced in E. Coli, HyTest,        Finland).    -   Maxisorp 96-well flat-bottomed microplate (Nunc, Denmark) primed        with monoclonal antibodies (hinge 76 antibody, for example, from        Bio-Rad) which recognises the proBNP(1-108) hinge sequence:        epitope RAPR₇₆S₇₇P (SEQ ID NO: 55) (Giuliani et al., Clin.        Chem., 52: 6, 1054-1061, 2006)    -   20G7 monoclonal antibody (Bio-Rad) coupled to peroxidase    -   24C5 monoclonal antibody (HyTest, Finland) coupled to peroxidase    -   26E2 monoclonal antibody (HyTest, Finland) coupled to peroxidase    -   PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets,        ref: 18912-014 (Invitrogen)    -   Tween®20 (Sigma-Aldrich, USA, #P1379)

10.2. Method:

10.2.1. Principle of the Analytical Assay of proBNP(1-108):

Initially, a standard range of 20 to 10,000 pg/ml of proBNP(1-108) wasprepared in a 0.1% PBS-T buffer from recombinant ProBNP(1-108). Theassay is based on the sandwich ELISA principle on a microplate, usingfor capture on a solid phase a monoclonal antibody (hinge 76 antibody,for example, from Bio-Rad), and recognising the hinge sequence ofproBNP(1-108): epitope RAPR₇₆S₇₇P (SEQ ID NO: 55) fixed by passiveadsorption by way of 100 μl of 0.5 μg/ml solution per well.

100 μl of monoclonal antibody solution (20G7, 24C5 or 26E2 antibodies)at a concentration of 0.5 μg/ml and coupled to peroxidase in solution in0.1% PBS-T buffer (containing 0.1% semi-skimmed milk) were used asdetection antibodies. Besides this technical point, the protocol wasidentical to that of the ELISA in Example 8. The detectioncharacteristics of 20G7 were thus compared with those of the 24C5 and26E2 monoclonal antibodies.

Table 5 shows the results of the analytical assay of proBNP(1-108) whichare expressed in optical density (OD) and were obtained by using saidantibodies in the presence of standard concentrations of proBNP(1-108).

10.3. Results:

TABLE 5 Analytical assay of proBNP(1-108) with different antibodies 24C5 26 E2 ProBNP(1-108) (pg/ml) 20 G7 (Hytest) (Hytest) 10,000 >4 0.2200.158 5,000 3.845 0.117 0.041 2,500 3.272 0.069 0.031 1,250 1.955 0.0680.024 625 0.997 0.070 0.024 312.5 0.512 0.035 0.028 156.25 0.264 0.0550.027 78 0.126 0.056 0.033 39 0.092 0.056 0.028 20 0.069 0.052 0.028 00.045 0.044 0.024

It is highly remarkable to note that the 20G7 antibody of the presentinvention detects not only BNP(1-32) but also proBNP(1-108). Moreover,in this case too, the two HyTest antibodies behave quite differentlyfrom the 20G7 antibody. This confirms the significant benefit of the20G7 antibody in BNP(1-32) and proBNP(1-108) assays.

FIG. 6 illustrates the linear standard range, obtained with the 20G7monoclonal antibody, of 20 to 10,000 pg/ml of proBNP (r²=0.99, FIG. 6),whilst the two commercial antibodies from Hytest are not very effectiveor not effective at all in detecting proBNP(1-108), even at highproBNP(1-108) concentrations (Table 5).

Example 11 ProBNP(1-108) and BNP(1-32) Assays Using Other MonoclonalAntibodies Obtained by the Inventors

Table 6 shows the results produced, in accordance with the two ELISAprotocols from examples 8 and 10, with the 11A8 and 17F10 monoclonalantibodies.

These labelled monoclonal antibodies used in detection are highlycapable of detecting BNP(1-32) and proBNP(1-108) when a rabbitpolyclonal (L21016) which targets the ¹SPKMV⁵ region or the hinge 76antibody respectively, are used for capture.

Table 6 shows the results of the analytical assays of proBNP(1-108) andBNP(1-32) which are expressed in optical density and were obtained bysaid antibodies in the presence of standard concentrations ofproBNP(1-108) and BNP(1-32) respectively.

TABLE 6 Analytical assay of proBNP(1-108) and BNP(1-32) with differentmonoclonal antibodies BNP(1-32) or proBNP(1- proBNP(1-108) BNP(1-32)108) (pg/ml) 17F10 11A8 17F10 11A8 10,000 3.769 3.734 3.717 3.693 50003.808 3.779 2.712 2.534 2,500 3.453 3.024 1.495 1.481 1,250 2.118 1.4060.828 0.871 625 0.937 0.618 0.341 0.389 312.5 0.489 0.259 0.172 0.211156 0.242 0.130 0.095 0.123 78 0.139 0.088 0.070 0.114 39 0.125 0.0870.059 0.097 20 0.100 0.078 0.059 0.071 0 0.076 0.063 0.059 0.068

It is highly remarkable to note that the 17F10 and 11A8 antibodies ofthe present invention detect not only BNP(1-32) but also proBNP(1-108).

Example 12 BNP(1-32) Assay and proBNP(1-108) Assay in Subjects withCongestive Heart Failure and in Normal Subjects 12.1. Samples:

55 EDTA plasmas from subjects with congestive heart failure, whobelonged to one of NYHA (New York Heart Association) classes I to IIIand had signed a voluntary consent form, originating from a commercialsource (ProMedex, NY, USA). The studied population is as follows: 10patients of NYHA class I, 21 patients of NYHA class II and 24 patientsof NYHA class III.

48 EDTA plasmas from normal subjects (healthy volunteers, ProMedex, NY,USA).

12.2. Materials and Methods for the BNP(1-32) and the proBNP(1-108)Assays

The materials and methods used were identical to those described abovein 8.2 for BNP(1-32), and to those described above in 10.2 for theproBNP(1-108).

12.3 Results

12.3.1. Results of the BNP(1-32) and the proBNP(1-108) Assays inPatients with Congestive Heart Failure

The BNP(1-32) values obtained from plasmas from patients with congestiveheart failure, by means of the BNP(1-32) assay disclosed in 8.2, werefound to correlate with those of the proBNP(1-108) assay according tothe invention (r²=0.935, FIG. 7).

More in detail, correlations are maintained when the patients arestudied according to their NYHA class (r²=0.997, r²=0.903, r²=0.832respectively for the NYHA classes I, II and III, FIG. 8 A, B et Crespectively).

Thus, these results with the 20G7 antibody confirm once more theusefulness of the BNP(1-32) or the proBNP(1-108) assay as a marker ofcongestive heart failure.

These experiments were reproduced with the 24C5 and 26E2 antibodies(HyTest) in a labelled form, but no correlation was observed.

12.3.2. Results of the BNP(1-32) and the proBNP(1-108) Assays in HealthySubjects

The proBNP(1-108) values obtained from plasmas from healthy subjects, bymeans of the proBNP(1-108) assay using the monoclonal hinge 76 antibodyin the solid phase and the 20G7 antibody-peroxidase conjugate fordetection, were found to be highly correlated (r²=0.702) with those ofthe BNP(1-32) assay using the 20G7 antibody in detection (FIG. 9).

In conclusion, it is therefore very clear that the 20G7 antibodyaccording to the invention is entirely appropriate for BNP(1-32) andproBNP(1-108) assays in patients suffering from congestive heartfailure, by detecting a higher amount of BNP(1-32) and proBNP(1-108) inpatients suffering from congestive heart failure than in healthysubjects (Table 7).

TABLE 7 proBNP(1-108) (pg/ml) BNP(1-32) (pg/ml) Healthy subjects 37 ± 32227 ± 172 NYHA patients 762 ± 839 1716 ± 1754

Example 13 BNP(1-32) and proBNP(1-108) Assays in Patients Suffering fromrenal failure 13.1. Samples:

EDTA plasmas from 33 patients with renal failure who has signed avoluntary consent form, originating from Lapeyronie hospital,Montpellier, France.

13.2. Materials and Methods for the BNP(1-32) and the proBNP(1-108)Assays

The materials and methods used were identical to those described abovein 8.2 for BNP(1-32) and in 10.2 for proBNP(1-108).

13.3 Results

The proBNP(1-108) values obtained from plasmas of patients suffering ofrenal failure, by means of the proBNP(1-108) assay using the hinge 76antibody in the solide phase and 20G7 antibody-peroxydase conjugate indetection, were found to be strongly correlated (r²=0.899) to those ofthe BNP(1-32) assay using the 20G7 antibody according to the invention(FIG. 10). The 20G7 antibody according to the invention is highlyappropriate for the BNP(1-32) and the proBNP(1-108) assay in patientswith renal failure.

Example 14 ProBNP(1-108) Assay in Patients with Ischemic Stroke

14.1. Patients samples:

32 citrated plasma samples from patients with ischemic stroke admittedto the Emergency Department within 3 hours of the Stroke onset weretested. The stroke severity was assessed by the National Institutes ofHealth Stroke Scale (NIHSS).

42 citrated plasma samples from apparently healthy blood donor matchedby gender and age with the patients from the Stroke population weretested. All the citrated plasma samples were stored at −80° C. Prior theanalysis, the samples were thawed and centrifuged at 3000 g for 15 minat 4° C.

14.2. Material and Method:

All the samples were tested with the BioPlex™ 2200 proBNP assay(Bio-Rad).

14.2.1. Principe of the Technology:

The BioPlex™ 2200 combines multiplex, magnetic bead and flow cytometrytechnologies to provide multi-analyte detection on a fully automatedrandom access platform. Magnetic particles (8 μm diameter,carboxyl-modified surface) are dyed with two fluorophores(classification dyes, CL1 and CL2) which emit at distinct wavelengthsand adsorb significantly at 635 nm. The reporter fluorophore,β-phycoerythrin (PE) was chosen for its high molar extinctioncoefficient, quantum yield, resistance to photobleaching, lack ofself-quenching and stability. The detector simultaneously measures lightat three wavelengths: the two classification dyes and the reporter dye.

14.2.2. BioPlex™ 2200 proBNP: Assay Principle

The BioPlex™ 2200 proBNP assay is a two-step sandwich fluorescenceimmunoassay. In a first step, the BioPlex™ 2200 system combines 50 μL ofpatient sample, magnetic dyed beads coated with the anti-proBNP(1-108)monoclonal antibody (hinge 76 monoclonal antibody recognizing theepitope RAPR₇₆S₇₇P (SEQ ID NO: 58), Bio-Rad) and assay buffer into areaction vessel. Then, after 11 minutes of incubation and wash cycles,the anti-human BNP monoclonal antibody 20G7 conjugated to phycoerythrin(PE) is added and incubated for 2 minutes. After removal of excessconjugate, the bead mixture is passed through the detector whichidentifies the dyed beads and the amount of antigens captured on thebeads by the fluorescence of PE. After calibration using a set of sixdistinct calibrators, the three levels of quality controls and patientsamples results are expressed in pg/mL.

Two Quality Control beads are also tested with each sample to enhancethe integrity of the overall system.

14.3. Results:

The distributions of the BioPlex™ 2200 proBNP values for the Control andthe ischemic stroke populations are shown in Table 8 and FIG. 11. Thelevel of proBNP(1-108) was significantly higher in the ischemic strokegroup compared to the control group (Mann-Whitney, p<0.0001). Theresults demonstrate that the proBNP(1-108) is also a useful plasmabiomarker for the early diagnosis of Ischemic stroke.

TABLE 8 BioPlex ™ 2200 proBNP concentrations in ischemic stroke andcontrol citrated plasma samples (minimum, 1^(st) quartile, median,3^(rd) quartile and maximum values). Minimum 1^(st) Quartile 3^(rd)Quartile Maximum Populations (pg/mL) (pg/mL) Median (pg/mL) (pg/mL)(pg/mL) Control population 0 0  1 2 23 (N = 42) (IC95%: 0-2) Ischemic 234 71 219 1019 Stroke population (IC95%: 38-145) (N = 32)

The inventors clearly demonstrate that the proBNP(1-108) sandwich assayusing the monoclonal antibody 20G7 described in this invention canmeasure proBNP(1-108) concentrations in patients with stroke.

Example 15 ProBNP(1-108) Assay in Patients Suffering from an AcuteCoronary Disorder 15.1. Samples:

EDTA plasmas from 27 patients with an acute coronary disorder (withTroponine I plasma mean values reaching 12.5±6.9 ng/ml) originating froma commercial source (ProMedex, NY, USA)

EDTA plasmas from 48 healthy subjects (healthy volunteers, ProMedex, NY,USA).

15.2. Materials and Methods

The materials and methods used for the BNP(1-32) assay were identical tothose described above in 8.2, and for the proBNP(1-108) in 10.2.

15.3 Results

The proBNP(1-108) values obtained from plasmas of patients admitted inemergency and diagnosed for an acute coronary disorder by means of theproBNP(1-108) assay described above, were found to be stronglycorrelated (r²=0.956) to those obtained with the BNP(1-32) assay usingthe 20G7 antibody according to the invention (FIG. 12). Levels ofBNP(1-32) and proBNP(1-108) of patients with an acute coronary disorder(668±619 and 1,518±1,533 pg/ml for proBNP(1-108) and BNP(1-32)respectively and assayed according to the invention) were higher thanthose of healthy subjects (37±32 and 227±172 pg/ml for proBNP(1-108) andBNP(1-32) respectively and assayed according to the invention).

It is therefore very clear that the sandwich assay using the 20G7antibody according to the invention (coupled to peroxidase) allowsproBNP(1-108) concentrations, which are proportional to the level ofBNP(1-32), to be measured. These results once again prove the usefulnessof assaying proBNP(1-108) and BNP(1-32) as markers, in particular asmarkers of an acute coronary disorder.

Example 16 Assay of proBNP(1-108) in the Glycosylated Form Using the20G7 Monoclonal Antibody 16.1. Materials:

-   -   proBNP(1-108) (recombinant protein produced in E. coli for the        non-glycosylated form, and from reprogrammed HEK293 cells for        the glycosylated form, HyTest, Finland)    -   Maxisorp™ 96-well flat-bottomed microplate (Nunc, Denmark)    -   PBS (phosphate buffered saline) buffer, pH 7.4, Gibco tablets,        ref: 18912-014 (Invitrogen)    -   Tweed® 20 (Sigma-Aldrich, USA, #P1379)    -   hinge 76 monoclonal antibody which recognises the hinge sequence        of proBNP(1-108): epitope RAPR₇₆S₇₇P (SEQ ID NO: 55) (Giuliani        et al., Clin. Chem., 52: 6, 1054-1061, 2006)    -   20G7 monoclonal antibody (Bio-Rad) coupled to peroxidase

16.2. Method:

16.2.1. Principle of the Analytical Assay of Glycosylated proBNP:

A standard range of 20 to 10,000 pg/ml of glycosylated proBNP(1-108) wasprepared in a 0.1% PBS-T buffer. A standard range of 20 to 10,000 pg/mlof non-glycosylated proBNP(1-108) was prepared in the same way in a 0.1%PBS-T buffer.

The assay is based on the sandwich ELISA principle on a microplate,using for capture on a solid phase the monoclonal antibody (hinge 76antibody, from Bio-Rad), recognising the epitopic sequence RAPR₇₆S₇₇P(SEQ ID NO: 55) of proBNP(1-108) and fixed by passive adsorption by wayof 100 μl of 0.5 μg/ml solution per well.

100 μl of a solution of the 20G7 monoclonal antibody according to theinvention coupled to peroxidase at a concentration of 0.5 μg/ml, insolution in 0.1% PBS-T containing 1% milk (semi-skimmed), were used asdetection reagents. The rest of the protocol was identical to that ofthe ELISA in Example 10. The detection characteristics of the 20G7antibody were thus compared for the two forms ofproBNP(1-108)-glycosylated and non-glycosylated.

16.3. Results:

The results shown in Table 9 and FIG. 13 correspond to the glycosylatedproBNP(1-108) and non-glycosylated proBNP(1-108) assays by means ofimmunoassay using the immobilised hinge 76 antibody and the 20G7antibody for detection.

TABLE 9 optical density values at 490 nm from the assay of eachproBNP(1-108) form tested. non- glycosylated glycosylated proBNP range(pg/ml) proBNP(1-108) proBNP(1-108) 10,000 4 3.833 5000 3.807 3.8012,500 3.829 3.023 1,250 3.256 1.679 625 1.816 0.925 312.5 0.989 0.465156 0.510 0.268 78 0.221 0.130 39 0.109 0.087 20 0.063 0.041 9 0.0370.028 0 0.021 0.023

ProBNP(1-108) can be detected just as well in the non-glycosylated formas in the glycosylated form by using 20G7. The signal/background ratiois slightly greater for non-glycosylated proBNP than for itsglycosylated form (signal/background ratios of 3 and 1.8 respectivelyare obtained at 20 pg/ml).

Example 17 Immunoreactivity of proBNP(1-108) in its Glycosylated Formand Implications for the proBNP(1-108) Assay with the Hinge 76 Antibody17.1. Materials:

ProteOn XPR36 analyser (surface plasmon resonance SPR technology,Bio-Rad, USA)

proBNP(1-108) (recombinant protein, HyTest, Finland) in the glycosylatedand non-glycosylated forms (13 to 200 nM)

anti Fc fragment antibodies (Sigma, USA)

monoclonal hinge 76 antibody (Bio-Rad) which recognises the epitopeRAPR₇₆S₇₇P (SEQ ID NO: 55) and is used at a concentration of 30 μg/ml in0.1% PBS-T

PBS buffer (phosphate buffered saline), pH 7.4

17.2. Method: 17.2.1. Principle:

The ProteOn XPR36 analyser (the principle of which is based on thesurface plasmon resonance technology (SPR)), was used to define thekinetics and the affinity of the interaction of the 20G7 monoclonalantibody and with glycosylated and non-glycosylated proBNP(1-108) forms.The inventors followed the manufacturer's instructions. A monoclonalantibody was immobilised on a biosensor (solid surface) by using an antiFc fragment antibody whilst the glycosylated or non-glycosylated solubleantigen proBNP(1-108) circulated at increasing concentrations (13 to 200nM) in a constant flow on the surface of the biosensor at roomtemperature. The angle at which the SPR signal is detected is directlyproportional to the refractive index of the medium in which theevanescent wave propagates. The variations in the refractive index areexpressed in resonance units (RU, where 1000 resonance units correspondto 1 ng of fixed proteins per mm² of surface area). The quantificationof the interactions and the affinity between the antigen and themonoclonal antibody is assessed by calculating the association rateconstant (ka) and the dissociation rate constant (kd) by global dataprocessing using the device software (Bio-Rad). The equilibriumdissociation constant (KD=kd/ka) in mol/l reflects the affinity of theglycosylated or non-glycosylated proBNP(1-108) antigen for themonoclonal antibody.

17.2.2. Results:

Table 10 shows the characteristics of the interaction between theanti-hinge antibody (hinge 76 antibody with the epitope RAPR₇₆S₇₇P (SEQID NO: 55) Bio-Rad) and glycosylated and non-glycosylated proBNP(1-108).Although the affinity constant between the anti-hinge antibody andnon-glycosylated proBNP(1-108) was greater (1.73.10⁻¹⁰) than that forglycosylated proBNP(1-108) (2.35.10⁻⁸), the antibody targeting the hingeregion of proBNP(1-108) recognised both the glycosylated andnon-glycosylated forms with high affinity.

TABLE 10 Reactivity of the hinge 76 antibody towards glycosylated andnon- glycosylated proBNP(1-108) Hinge 76 antibody ka (M⁻¹s⁻¹) kd (s⁻¹)KD = kd/ka M non-glycosylated 1.12 · 10⁶ 1.94 · 10⁻⁴ 1.73 · 10⁻¹⁰proBNP(1-108) glycosylated proBNP(1- 1.17 · 10⁵ 2.72 · 10⁻³ 2.35 · 10⁻⁸108)

Example 18 Biepitopic and Triepitopic Calibrators

18.1. The Structure of all of the Biepitopic and Triepitopic CalibratorsAccording to the Invention May be Linear or Branched, Provided that theImmunoreactivity of the Incorporated Epitopes is Preserved.

The synthesis protocols which may be used to produce these calibratorsare those in the field of organic chemistry of peptides well known tothe one skilled in the art (in this context, see “Peptide synthesis” inExample 1).

For the epitopes E₂ and E₃, the linear peptide sequences according tothe invention may be selected in a non-limiting manner from the groupconsisting of the following sequences:

SEQ ID NO: 56: PRSPKMVQG SEQ ID NO: 57: APRSPKMV SEQ ID NO: 58: SGLGCKLVSEQ ID NO: 59: SPKMVQGSG SEQ ID NO: 60: YTLRAPRSPKMVG

18.2. Examples of Synthesised Epitopes According to the Invention

The inventors synthesised the following calibrators:

18.2.1. Biepitopic Calibrators: CaliproBNP1:Ac-YTLRAPRSPKMV-Ahx-SFGRKMDRISS-CONH₂

CaliproBNP2: Ac-YTLRAPRSPKMV-Ahx-CFGRKMDRISSSSGLGCK-CONH₂

CaliProBNP3: Ac-YTLRAPRSPKMVQG-Ahx-FGRKMDR-CONH₂

These three biepitopic calibrators can be used to calibrate aproBNP(1-108) assay, as described above in 10.2, based on theimmobilisation in a solid phase of the monoclonal hinge 76 antibody thatrecognizes the RAPRSP (SEQ ID NO: 55) (Giuliani et al., supra) and, indetection the monoclonal 20G7 antibody-peroxydase for example.

CaliproBNP4: Ac-FGRKMDR-Ahx-SGLGC*KVLRRH-COOH CaliproBNP4b:Ac-FGRKMDR-Ahx-SGLGC*KVLR-CONH₂

These two biepitopic calibrators can be used to calibrate a BNP(1-32)assay, based on the immobilisation in a solid phase of the monoclonalantibody directed the C-terminal portion of BNP(1-32), such as forexample monoclonal antibodies 50B7 or 50E1 (HyTest, Finland) and, indetection the monoclonal 20G7 antibody-peroxydase for example.

CaliproBNP5: Ac-SPKMVQGSG-Ahx-FGRKMDR-CONH₂

This biepitopic calibrator can be used to calibrate a BNP(1-32) assay,as described above in 8.2, based on the immobilization in a solid phaseof the polyclonal antibody L21016 (Bio-Rad) and, in detection themonoclonal 20G7 antibody-peroxydase for example.

In the calibrators sequences described above, the structural formula ofthe Ahx group is NH—(CH₂)₅—CO.

The binding group of formula —NH—(CH₂)₅—CO— is derived from a well-knowncoupling agent, amino-hexanoic acid (AHX), which enables to covalentlycouple two peptide sequences together.

C*=C(Acm)=Cysteine blocked by an acetamidomethyl (protecting group,well-known from the one skilled in the art).

18.2.2. Triepitopic Calibrators: CaliproBN P6:Ac-YTLRAPRSPKMV-Ahx-FGRKMDR-Ahx-SGLGC*KVLRRH-COON CAliproBNP6b:Ac-YTLRAPRSPKMV-Ahx-FGRKMDR-Ahx-SGLGC*KVLR-CONH₂

These two triepitopic calibrators can be used to calibrate both aproBNP(1-108) and a BNP(1-32) assay, using antibodies such as themonoclonal hinge 76 antibody that recognizes the sequence RAPRSP (SEQ IDNO: 55) (Giuliani et al., supra) and the monoclonal 20G7 antibody of theinvention and an antibody with an epitope directed against theC-terminal portion of BNP(1-32) for example.

18.3. Materials and Methods

To depict the usefulness of these calibrators, results of calibrationand stabilities are displayed in two different formats, BioPlex™ assayand ELISA assay.

-   -   The proBNP(1-108) biepitopes, the CaliproBNP1 and CaliproBNP3        compounds, were tested by means of the BioPlex™ 2200 proBNP        assay as described in Example 14.    -   The BNP(1-32) biepitope, the CaliproBNP5 compound, was tested by        means of the assay described in Example 8.2 and the triepitope,        the CaliproBNP6 compound was tested by means of the assay        described in Example 10.2.

18.4. Protocole

The different compounds were tested at different concentrations dilutedin 0.1 M succinate buffer, pH 7.6, containing 5% BSA, 2 mM CaCl₂, 10%antiproteases cocktail (Sigma reference P2714), 0.1% Proclin, 0.095%NaN₃ and 0.1% sodium benzoate.

The CaliproBNP1 calibrator was diluted to 1 μg/mL, 0.2 μg/mL, 0.1 μg/mL,and 0.02 μg/mL, the CaliproBNP3 calibrator was diluted to 1.6 μg/mL, 0.8μg/mL, 0.3 μg/mL, 0.06 μg/mL, the CaliproBNP5 and CaliproBNP6calibrators were diluted to from 100 ng/ml to 0.01 ng/ml in the same 0.1M succinate buffer, pH 7.6.

The compounds stability was studied in accelerated condition (roomtemperature) compared to synthetic BNP(1-32) (Sigma-Aldrich, Etats-Unis,#B-5900) and recombinant proBNP(1-108) (HyTest Ref. 8PRO8) in thefollowing way:

Recombinant proBNP(1-108), synthetic BNP(1-32) and calibrator peptideswere diluted in 0.1 M succinate buffer, pH 7.6 described above. Eachsolution was divided in 10 tubes placed at room temperature (20° C.±5°C.). Then one tube of each solution was freezed at J0, J+7, J-F14, J+21.At J+21, the different solutions were thawed and assayed by means of theBioPlex™ 2200 proBNP assay described in Example 14 or of theimmunoassays described in Examples 8.2 and 10.2

In the ELISA assay, a range of proBNP(1-108), BNP(1-32) and CaliproBNP6from 25 ng/mL to 0.04 ng/mL was tested. For CaliBNP4 and CaliBNP5, thetested range was from 2 ng/mL to 0.004 ng/mL.

In the BioPlex™ 2200 proBNP assay, the stability of two concentrationsof proBNP(1-108), 10 ng/mL and 1 ng/mL, of CaliproBNP1, 2 μg/mL and0.125 μg/mL and CaliproBNP3, 1 μg/mL and 0.3 μg/mL, were analyzed. InELISA format, three concentrations of proBNP(1-108) and BNP(1-32), 1.56ng/mL, 0.78 ng/ml and 0.39 ng/mL, and of CaliproBNP5, 62.5 pg/mL, 31ng/mL and 15.6 ng/mL, were analysed.

18.5. Results

18.5.1 CaliproBNP1 and CaliproBNP3 Calibrators in the Bioplex™ 2200proBNP Assay

The results of the assay of the CaliproBNP1 and CaliproBNP3 compounds inrange of different concentrations are displayed in FIGS. 14 and 15respectively.

In a BioPlex™ 2200 proBNP assay, the CaliproBNP1 and CaliproBNP3compounds enable to generate increasing signal with the compoundconcentration. These compounds are therefore usable as calibrators ofthe proBNP(1-108) assay, once standardized on the proBNP(1-108)molecule.

The results of the accelerated stability test of the recombinantproBNP(1-108) and the CaliproBNP1 and CaliproBNP3 compounds in range ofdifferent concentrations are displayed in Table 11.

TABLE 11 Liquid stability* at room temperature** in buffer*** expressedby the ratio “signal at Concentration J0 + X/signal at J0” Compound(μg/mL) J0 + 7 J0 + 14 J0 + 21 recombinant 0.001 0.79 0.65 0.51proBNP(1-108) 0.01 0.85 0.72 0.69 CaliproBNP1 0.125 0.93 0.92 0.95 20.95 0.93 0.91 CaliproBNP3 0.3 0.91 0.98 1.15 1 0.92 1.02 1.06 *Norme ofacceptance of stabilities: to make the stability at day X after J0accetable, the ratio signal at J0 + X/signal at J0 has to be equal to1.00 ± 0.2. **Room temperature: 20° C. ± 5° C. ***0.1 M succinatebuffer, pH 7.6, containing 5% BSA, 2 mM CaCl₂, 10% antiproteasescocktail (Sigma reference P2714), 0.1% Proclin, 0.095% NaN3 and 0.1%.sodium benzoate.

Biepitopic calibrators CaliproBNP1 and CaliproBNP3 clearly show a higherstability than recombinant proBNP(1-108).

18.5.2 CaliproBNP5 and CaliproBNP6 calibrators in immunoassays based onthe use of 20G7 antibody

The results of the test of the CaliproBNP5 and CaliproBNP6 compounds inrange of different concentrations are displayed in FIGS. 16 and 17,respectively.

In the BNP(1-32) and the proBNP(1-108) assay, the biepitopic compoundCaliproBNP5 and the triepitopic compound CaliproBNP6 enable to generatea signal increasing with the compound concentration. These compounds aretherefore usable as calibrators of the proBNP(1-108) and the BNP(1-32)assay.

The results of the accelerated stability assay of recombinantproBNP(1-108), BNP(1-32) and the CaliproBNP5 compound, in range ofdifferent concentrations are displayed in Table 12.

TABLE 12 Liquid stability* at room temperature** in buffer*** expressedby the ratio “signal at Concentration J0 + X/signal at J0” Compound(μg/mL) J0 + 7 J0 + 14 J0 + 21 recombinant 1.56 0.58 0.5 0.45proBNP(1-108) 0.78 1.08 0.82 0.88 0.39 0.71 0.66 0.79 synthetic 1.560.71 0.7 0.55 BNP(1-32) 0.78 0.69 0.65 0.53 0.39 0.81 0.7 0.53CaliproBNP5 0.0625 1.02 1.02 1.1 0.031 1.06 1.01 1.06 0.0156 1.1 1.011.10 *Norme of acceptance of stabilities: to make the stability at day Xafter J0 accetable, the ratio signal at J0 + X/signal at J0 has to beequal to 1.00 ± 0.2. **Room temperature: 20° C. ± 5° C. ***0.1 Msuccinate buffer, pH 7.6, containing 5% BSA, 2 mM CaCl₂, 10%antiproteases cocktail (Sigma reference P2714), 0.1% Proclin, 0.095%NaN3 and 0.1%. sodium benzoate.

Once again, the CaliproBNP5 biepitopic calibrator displays a higherstability than recombinant proBNP(1-108) and BNP(1-32)

Summary table of the sequences SEQ ID NO: SEQUENCES 1HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSP KMVQGSGCFGRKMDRISSSSGLGCKVLRRH 2SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH 3HPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPR 4 TGCFGRKMDRISTSTAIGCKVL 5SGCYGRKMDRISTSTAIGCKVL 6 SGCFGRKMDRISSSSGLGCKVL 7 SGCFGRKMDRIATSTAIGCKVL8 FGRKMDR 9 GRKMDR 10 FGRKMD 11 RKMDRI 12 SPKMVQGSGCFGRKM 13KMVQGSGCFGRKMDR 14 VQGSGCFGRKMDRIS 15 GSGCFGRKMDRISSS 16 GCFGRKMDRISSSSG17 FGRKMDRISSSSGLG 18 RKMDRISSSSGLGCK 19 MDRISSSSGLGCKVL 20RISSSSGLGCKVLRR 21 VQGSGCFGR 22 SPKMVQGSGC 23 MDRISSSSGLG 24 RKMDRI 25RKMDRISS 26 SPKMVQGSGC 27 PKMVQGSGCF 28 KMVQGSGCFG 29 MVQGSGCFGR 30VQGSGCFGRK 31 QGSGCFGRKM 32 GSGCFGRKMD 33 SGCFGRKMDR 34 GCFGRKMDRI 35CFGRKMDRIS 36 FGRKMDRISS 37 GRKMDRISSS 38 RKMDRISSSS 39 KMDRISSSSG 40MDRISSSSGL 41 DRISSSSGLG 42 RISSSSGLGC 43 ISSSSGLGCK 44 SSSSGLGCKV 45SSSGLGCKVL 46 SSGLGCKVLR 47 SGLGCKVLRR 48 GLGCKVLRRH 49 GCFGRKM 50CFGRKMD 51 FGRKMDR 52 GRKMDRI 53 RKMDRIS 54 SPKMV 55 RAPRSP 56 PRSPKMVQG57 APRSPKMV 58 SGLGCKVL 59 SPKMVQGSG 60 YTLRAPRSPKMVG 61 FGRKMDRISSSS 62AGRKMDR 63 GCFGRKMDRIS 64 SFGRKMDRISS 65 CFGRKMDRISSSSGLGCK 66YTLRAPRSPKMV 67 YTLRAPRSPKMVQG 68 SGLGCKVLRRH 69 SGLGCKVLR

1-29. (canceled)
 30. A polypeptide carrying a human BNP(1-32) epitopehaving the formula (I):a₁-R₁-X₁-FGRKMDR-X₂-R₂-a₂  (I) wherein a₁ may be H or represent afunctional group or chemical group selected from a thiol, alcohol,aminoxy, primary amine or secondary amine functional group, an aminocarboxyl group, a biotinyl group and an acetyl group; a₂ may representan OH, NH₂ functional group or an alcoxyl group; X₁ is absent or presentand when present is selected among C and GC; X₂ is absent or present andwhen present is selected among I and IS; R₁ and R₂, which may be thesame or different, present or absent, represent any amino acid or apeptide chain of 2 to 15 amino acids, provided that said polypeptide offormula (I) does not include any portion of human BNP(1-32) of more than11 amino acids including the sequence GCFGRKMDRIS.
 31. The polypeptideaccording to claim 30, said polypeptide being selected in the groupconsisting of a₁-SGCFGRKMDR-a₂ (SEQ ID NO: 33), a₁-GCFGRKMDRI-a₂ (SEQ IDNO: 34), a₁-CFGRKMDRIS-a₂ (SEQ ID NO: 35) and a₁-FGRKMDRISS-a₂ (SEQ IDNO: 36), wherein a₁ and a₂ are as defined in claim
 1. 32. Thepolypeptide according to claim 30, having the formula (II):a₁-FGRKMDR-a₂  (II) wherein a₁ and a₂ are as defined in claim
 1. 33. Amethod for preparing ligands directed against human BNP(1-32) or humanproBNP(1-108) as well as the respective fragments thereof comprising thesequence FGRKMDR, wherein the ligands are elicited against a polypeptidecarrying a human BNP(1-32) epitope having the formula (I):a₁-R₁-X₁-FGRKMDR-X₂-R₂-a₂  (I) wherein a₁ may be H or represent afunctional group or chemical group selected from a thiol, alcohol,aminoxy, primary amine or secondary amine functional group, an aminocarboxyl group, a biotinyl group and an acetyl group; a₂ may representan OH, NH₂ functional group or an alcoxyl group; X₁ is absent or presentand when present is selected among C and GC; X₂ is absent or present andwhen present is selected among I and IS; R₁ and R₂, which may be thesame or different, present or absent, represent any amino acid or apeptide chain of 2 to 15 amino acids, provided that said polypeptide offormula (I) does not include any portion of human BNP(1-32) of more than11 amino acids including the sequence GCFGRKMDRIS.
 34. A method forpreparing antibodies directed against human BNP(1-32) or humanproBNP(1-108) as well as the respective fragments thereof comprising thesequence FGRKMDR, comprising injecting an animal with a polypeptidecarrying a human BNP(1-32) epitope having the formula (I):a₁-R₁-X₁-FGRKMDR-X₂-R₂-a₂  (I) wherein a₁ may be H or represent afunctional group or chemical group selected from a thiol, alcohol,aminoxy, primary amine or secondary amine functional group, an aminocarboxyl group, a biotinyl group and an acetyl group; a₂ may representan OH, NH₂ functional group or an alcoxyl group; X₁ is absent or presentand when present is selected among C and GC; X₂ is absent or present andwhen present is selected among I and IS; R₁ and R₂, which may be thesame or different, present or absent, represent any amino acid or apeptide chain of 2 to 15 amino acids, provided that said polypeptide offormula (I) does not include any portion of human BNP(1-32) of more than11 amino acids including the sequence GCFGRKMDRIS.
 35. A method forpreparing a hybridoma which secretes a monoclonal antibody directedagainst human BNP(1-32) or human proBNP(1-108) as well as the respectivefragments thereof comprising the sequence FGRKMDR, wherein: samples oflymphocytes secreting immunoglobulins are taken from an animal immunisedwith a polypeptide according to claim 30, the lymphocytes are fused withmyeloma cells; in order to obtain a hybridoma.
 36. A hybridomaobtainable from the method defined in claim
 35. 37. A hybridomadeposited on Apr. 13, 2007 at the CNCM (Collection Nationale de Culturesde Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75 724Paris Cedex 15, France) under registration number CNCM I-3746.
 38. Aligand specific of an epitope of the sequence FGRKMDR.
 39. The ligandaccording to claim 38, selected from the group constituted by anantibody or a fragment of said antibody which recognises the epitope, anaptamer, and a polypeptide which specifically recognises the epitopeobtainable by phage display.
 40. The ligand according to claim 38,constituted by an antibody which specifically recognises an epitope ofthe sequence FGRKMDR, or a fragment of said antibody which specificallyrecognises the epitope.
 41. The ligand according to claim 38,constituted by an antibody which specifically recognises an epitope ofthe sequence FGRKMDR, or a fragment of said antibody which specificallyrecognises the epitope, wherein the antibody is a monoclonal antibody.42. A ligand specific of an epitope of the sequence FGRKMDR, constitutedby an antibody which specifically recognises an epitope of the sequenceFGRKMDR, or a fragment of said antibody which specifically recognisesthe epitope, wherein the antibody is a monoclonal antibody that isproduced by the hybridoma according to claim
 36. 43. A ligand specificof an epitope of the sequence FGRKMDR, constituted by an antibody whichspecifically recognises an epitope of the sequence FGRKMDR, or afragment of said antibody which specifically recognises the epitope,wherein the antibody is a monoclonal antibody that is produced by thehybridoma according to claim
 37. 44. The ligand according to claim 38,constituted by the monoclonal antibody produced by the hybridomadeposited on Apr. 13, 2007 at the CNCM (Collection Nationale de Culturesde Microorganismes, Institut Pasteur, 25, rue du Docteur Roux, 75 724Paris Cedex 15, France) under registration number CNCM I-3746.
 45. Theligand according to the specific of an epitope of the sequence FGRKMDR,harbouring at least one Complementary Determining Region (CDR) of theligand as defined in claim
 43. 46. A method for detecting, in abiological sample, human BNP(1-32) or a derivative of humanproBNP(1-108) containing the sequence FGRKMDR, comprising: 1) contactingthe biological sample with at least one ligand specific of an epitope ofthe sequence FGRKMDR under conditions allowing the formation ofantigen-ligand complexes, and 2) detecting any complex which may haveformed.
 47. The method according to claim 45, comprising at least oneadditional step of contacting the biological sample with at least oneadditional ligand specific of human BNP(1-32), human proBNP(1-108) orthe respective fragments thereof, and having a different specificityfrom that of the ligand specific of an epitope of the sequence FGRKMDR.48. The method according to claim 46, wherein the additional ligand isan antibody.
 49. A method of diagnosis, prognosis, risk stratificationor therapeutic follow-up of at least one cardiac and/or vascularpathology in an individual, comprising the following steps of: 1)contacting a biological sample from the individual with at least oneligand specific of an epitope of the sequence FGRKMDR under conditionsallowing the formation of antigen-ligand complexes, 2) detecting anycomplex which may have formed, and, 3) based on the result of thedetection in step 2, determining a diagnosis, a prognosis, a risk of thedevelopment or therapeutic follow-up of the pathology in the individual.50. The method according to claim 48, comprising at least one additionalstep of contacting the biological sample with at least one additionalligand specific of human BNP(1-32), human proBNP(1-108) or therespective fragments thereof, and having a different specificity fromthat of the ligand specific of an epitope of the sequence FGRKMDR. 51.The method according to claim 49, wherein the additional ligand is anantibody.
 52. The method according to claim 48, wherein the pathology isselected from the group comprising: congestive heart failure, acutecoronary syndrome, cerebrovascular accident, kidney failure, dyspnea,high blood pressure, atheromatous plaque rupture, patent ductusarteriosus in premature newborns, and/or diabetis.
 53. A multiepitopiccalibrator having the following general formula (III):t₁-E₁-L₁-E₂[-L_(k-1)-E_(k)]_(n)-t₂  (III) wherein: n is an integerbetween 0 and 8; k is an integer between 3 and n+2 when n>0; E₁, E₂, andE_(k) are different from one another, one representing aR₁-X₁-FGRKMDR-X₂-R₂ peptide sequence, wherein X₁ is absent or presentand when present is selected among C and GC; X₂ is absent or present andwhen present is selected among I and IS; R₁ and R₂, which may be thesame or different, present or absent, represent any amino acid or apeptide chain of 2 to 15 amino acids, provided that said polypeptide offormula (III) does not include any portion of human BNP(1-32) of morethan 11 amino acids including the sequence GCFGRKMDRIS, and the othersrepresenting a sequence of 3 to 15 amino acids selected from thesequence of human proBNP(1-108); t₁ represents a hydrogen atom, anacetyl group, a peptide sequence of 1 to 10 amino acids, a peptidesequence of 1 to 10 N-α acetylated amino acids, a biotinyl or biocytinylgroup, a peptide sequence of 1 to 10 amino acids carrying a biotinyl orbiocytinyl radical, or a linear amino alkyl (C₁-C₁₀) carbonyl chain; t₂represents a hydroxyl radical, an amino radical, a peptide sequence of 1to 10 amino acids, a peptide sequence of 1 to 10 amino acids carrying aterminal amino group, or a linear or branched amino alkyl (C₁-C₁₀)carbonyl chain; L₁ and L_(k), which may be the same or different,represent a binding group of peptide chains.
 54. The multiepitopiccalibrator according to claim 52, corresponding to the following generalformula (IV):t₁-E₁-L₁-E₂-t₂  (V) wherein E₁, E₂, L₁, t₁ and t₂ are as defined inclaim
 25. 55. The multiepitopic calibrator according to claim 52,wherein L₁ and L₂ represent:—NH—(CH₂)₅—CO—.
 56. The multiepitopic calibrator according to claim 54,selected from the group consisting of the multiepitopic calibratorsdefined by the following formulae:Ac-YTLRAPRSPKMV-L₁-SFGRKMDRISS-NH₂;Ac-YTLRAPRSPKMV-L₁-CFGRKMDRISSSSGLGCK-NH₂;Ac-YTLRAPRSPKMVQG-L₁-FGRKMDR-NH₂;Ac-FGRKMDR-L₁-SGLGC*KVLRRH-OH;Ac-FGRKMDR-L₁-SGLGC*KVLR-NH₂;Ac-SPKMVQGSG-L₁-FGRKMDR-NH₂;Ac-YTLRAPRSPKMV-L₁-FGRKMDR-L₂-SGLGC*KVLRRH-OH;and Ac-YTLRAPRSPKMV-L₁-FGRKMDR-L₂-SGLGC*KVLR-NH₂; wherein Ac representsan acetyl group and C* represents an acetamidomethyl-blocked cysteine.57. A kit for detecting human BNP(1-32) or human proBNP(1-108) as wellas the respective fragments thereof comprising the sequence FGRKMDR,comprising at least: (a) a ligand ligand specific of an epitope of thesequence FGRKMDR.; and (b) a multiepitopic calibrator having thefollowing general formula (III):t₁-E₁-L₁-E₂[-L_(k-1)-E_(k)]_(n)-t₂  (III) wherein: n is an integerbetween 0 and 8; k is an integer between 3 and n+2 when n>0; E₁, E₂, andE_(k) are different from one another, one representing aR₁-X₁-FGRKMDR-X₂-R₂ peptide sequence, wherein X₁ is absent or presentand when present is selected among C and GC; X₂ is absent or present andwhen present is selected among I and IS; R₁ and R₂, which may be thesame or different, present or absent, represent any amino acid or apeptide chain of 2 to 15 amino acids, provided that said polypeptide offormula (III) does not include any portion of human BNP(1-32) of morethan 11 amino acids including the sequence GCFGRKMDRIS, and the othersrepresenting a sequence of 3 to 15 amino acids selected from thesequence of human proBNP(1-108); t₁ represents a hydrogen atom, anacetyl group, a peptide sequence of 1 to 10 amino acids, a peptidesequence of 1 to 10 N-α acetylated amino acids, a biotinyl or biocytinylgroup, a peptide sequence of 1 to 10 amino acids carrying a biotinyl orbiocytinyl radical, or a linear amino alkyl (C₁-C₁₀) carbonyl chain; t₂represents a hydroxyl radical, an amino radical, a peptide sequence of 1to 10 amino acids, a peptide sequence of 1 to 10 amino acids carrying aterminal amino group, or a linear or branched amino alkyl (C₁-C₁₀)carbonyl chain; L₁ and L_(k), which may be the same or different,represent a binding group of peptide chains.
 58. A method of diagnosisof stroke in an individual comprising the following steps of: 1)contacting a biological sample from the individual with at least oneligand specific of an epitope of the sequence FGRKMDR under conditionsallowing the formation of antigen-ligand complexes, 2) detecting anycomplex which may have formed, and, 3) based on the results of thedetection in step 2, determining a diagnosis of stroke in theindividual; wherein the at least one ligand specific of an epitope ofthe sequence FGRKMDR of step 1) is used immobilised on a solid phase incombination with a monoclonal antibody directed against the RAPR₇₆S₇₇Psequence of proBNP(1-108) present in a labelled form, or the at leastone ligand specific of an epitope of the sequence FGRKMDR of step 1) isused in a labelled form in combination with a monoclonal antibodydirected against the RAPR₇₆S₇₇P sequence of proBNP(1-108) present in animmobilised form on a solid phase.